5-hydroxytryptamine stimulates cyclic AMP formation in the tunica muscularis mucosae of the rat oesophagus via 5-HT4 receptors

European Journal of Pharmacology, 211 (1992) 117-120 ct; 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.[8)

117

EJP 20997

Short communication

5-Hydroxytryptamine stimulates cyclic AMP formation in the tunica muscularis mucosae of the rat oesophagus via 5-HT 4 receptors A . P . D . W . F o r d , G.S. Baxter, R.M. E g l c n and D.E. Clarke Institute of Pharmacology, Syntt~ Research, 3401 l tilh'iew Ate, Palo Alto, CA 94304, U.S.A. Received 15 August 1991. revised MS received 27 November 1991. accepted 3 December 1991

The nature of 5-HT4 receptor coupling in the tunica muscularis mucosae of the rat oesophagus has bccn studied. 5-HT and renzapride stimulated cyclic AMP formation concentration dependently, with -log ECs, values of 7.1 and 6.8, respectively. Renzapride, relative to 5-HT, acted as a partial agonist. Tropisetron (ICS 205 930) and a novel 5-HT4 antagonist, SDZ 205 557, inhibited 5-HT-induced cyclic AMP production competitively, with pA 2 cstimates of 6.7 and 7.7, respectively. These data are consistent with the hypothesis that 5-HT4 receptors mediate relaxation of the smooth muscle cells of the tunica muscularis mucosae of rat oesophagus via activation of adenylyl cyclase. Oesophagus (rat); 5-HT (5-hydroxytryptaminc, serotonin); 5-HT4 receptors; cAMP; Benzamides (substituted); Tropisetron

1. Introduction

Recent studies have identified and characterised pharmacologically a 5-HT 4 receptor on the smooth muscle cells of the tunica muscalaris mucosae of the rat oesophagus (Baxter et al., 1991; Reeves et al., 1991). Activation of the receptor functions to inhibit tension development. However, the second messenger mediating 5-HT4-induced relaxation of smooth muscle is unknown. Thus, the present study was undertaken to determine whether cyclic AMP is involved, particularly as 5-HT 4 receptors have been shown to be linked positively to adenylyl cyclase in the central nervous system (Dumuis et al., 1988) and in myocardial cells (Kaumann et al., 1990). A preliminary account of the work was presented to the British Pharmacological Society, Glasgow meeting (Ford et al., 19911.

2. Materials and methods

2.1. Assay procedure The formation of cyclic AMP in rat oesophageal tunica muscularis mucosae was measured following the

Correspondence to: A.P.D.W. Ford, Institute of Pharmacology, Syntex Research, 3401 ltillview Ave, Palo Alto, CA 94304, U.S.A. Tel. 1.415.354 2413, fax 1.415.354 7400.

incorporation of [3H]adenine. Oesophagi from Sprague-Dawley rats (350-500 g; eight oesophagi were pooled for each individual experiment) were dissected free of the propria and the underlying tunica muscularis mucosae cross-chopped (Mcllwain tissue chopper, 350 ~m). The slices were placed in Tyrodc solution and digested with collagenase (type la; 2.5 m g / m l ) for 20 min. Subsequently, the slices were suspended in Tyrode solution containing cocaine (30 /xM), corticosterone (30/xM) and methysergide (1 /xM), gassed with O2-CO2 at 37°C (30 min equilibration), and were incubated with [3H]adenine (2 ~ C i / m l ) for 40 min at 37°C. After washing with Tyrode solution (3-4 times), 100 /xl aliquots of stirred slices were pipetted into minivials containing 3 - i s o b u t y i - l - m e t h y l x a n t h i n e (IBMX, 1 mM), antagonist or vehicle, and buffer (to 29(1 ~zl), and were shaken at 37°C for 30 min. Agonist (5-HT or renzapride) or vehicle (10 p.I) was then added for 15 min, after which time the reaction was terminated by the addition of 30 ~zl ice-cold HCI (2.2 M). Approximately 20(10 cpm [32P]cAMP was added to each assay tube to estimate recovery of the nucleotide. The [3H]cyclic AMP formed was extracted and isolated on a single column of acidic alumina using a method similar to that described by Alvarez and Daniels (1990). Radioactivity in the final [3H]cyclic AMP-containing eluate was determined with a liquid scintillation counter for both 3H and 32p. [3H]cyclic AMP counts were normalized to account for tissue mass, as reflccted by total 3H incorporated pcr assay vial.

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2.2. Data analysis

ICN (lrvine, CA). Acidic alumina (activity grade 1) was purchased from 1CN GmbH (Eschwege, F.R.G.). The remaining chemicals wcre obtained from Sigma Chemical Company (St. Louis, MO).

Data are expressed as cpm [3H]cyclic AMP per assay vial. Individual experiments were performed in quadruplicate; n refers to the number of individual experiments performed for each part of the study. Concentration-effect curves for 5-HT were expressed as a percentage stimulation above basal levels, and concentration-inhibition curves for tropisetron or SDZ 205 557 as a percentage of the rcsponse to 5-HT alone. K~ estimates for each antagonist were estimated from K i = IC5o/(i + [A]/EC~o), where [A] denotes concentration of agonist used and ICso denotes concentration of antagonist yielding a response equivalent to 50% of that achieved by agonist alone. Estimates for antagonist pA 2 were made by comparing agonist ECso values in the absence and presence (EC~ 0) of a single concentration ([B]) of antagonist (CR = EC.~dECso), such that pA 2 = - l o g ( [ B ] / ( C R - 1)).

3. Results Figure 1 shows that 5-HT and renzapride stimulated thc production of [3H] cyclic AMP in slices of rat oesophageal tunica muscularis mucoasc. The stimulation was concentration-dcpendent, to maxima of approximatcly 160% and 90% above basal levels (cpm/vial, basals: mean + S.E.M., 1773 + 53, 5-HT; 1123 + 74, renzapridc). Potencies for 5-HT and renzapridc (expressed as - l o g E C s 0 ) w e r e 7.1 +0.1 and 6.8 +_ 0.2, respcctivcly (mean + S.E.M., n = 3). Renzapride behaved as a partial agonist relative to 5-HT, with an intrinsic activity of approximately 0.5. Cyclic AMP formation induced by 5-HT and renzapride (each at 1 p.M) was inhibited concentration dependently by tropisetron, with an agonist-indepcndent IC5, value of approximately 3 p.M. Itcratively-fitted concentration-inhibition curves for tropisetron and SDZ 205 557 vcrsus 5-HT (1 IzM) are shown in fig. 2a. Values of - l o g IC5o for tropisetron and SDZ 205 557, interpolated from thc curves, are 5.6 + 0.2 (n = 4) and 6.3 + 0.2 (n = 3), respectively, yielding estimates of - l o g K i of 6.6 and 7.4. The surmountable nature of the antagonism produced by

2.3. Chemicals Rcnzapride, methysergide and SDZ 205 557 (2-diethylaminoethyl-(2-methoxy-4-amino-5-chloro)-benzoatc hydrochloride) were synthesiscd in the Institute of Organic Chemistry, Syntex Research. Tropisetron (ICS 205 930) was from Research Biochemicals Inc. (Natick, MA). [2,8-3H]Adenine (21.4 C i / m m o l was obtained from Du Pont-NEN (Boston, M A ) a n d [32p]adenosineY :5'-cyclic monophosphate (90.7 C i / m m o l ) was from

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Fig. 1. Stimulation of cyclic A M P formation by 5-HT (top) and renzapride (bottom), and inhibition of the response to each agonist (single 1 p.M concentration; approximate EC80) by tropisetron (ICS 205 930) in rat oesophageal tunica museularis mucosae. Data shown are means + S.E.M. from a representative quadruplicate study, which was performed three times. Basal values were (cpm/vial) 1773+53 (5-HT) and 1123_+74 (renzapride).

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[5-HT], M Fig. 2. (a) lteratively fitted concentration-inhibition curves tot tropisetron (ICS 205 930, II) and SDZ 205 557 (e) versus 5-HT (I ~M)-stimulated cyclic AMP formation in rat oesophageal tunica muscularis mucosae. Data are means+S.E.M. (n = 3-4 quadruplicate experiments), expressed as a percentage of the response to 5-|IT alone. (b,c) iteratively fitted concentration-effect curves for 5-HT-stimulated cyclic AMP formation in the absence ( ~ , o ) and presence of 3 /.tM tropisetron (ICS 205 93(I, II) or SDZ 205 557 (o) in rat oesophageal tunica muscularis mucosae. Data are means_-I: S.E.M. (n = 3 quadruplicate experiments), expressed as a percentage stimulation above basal levels.

each antagonist is shown in fig. 2b,c. Both compounds (at 3 p.M) produced parallel rightward displaccments of concentration-effcct curves for 5-HT. pA 2 estimates of 6.7 + 0.3 for tropisetron and 7.7 + 0.2 for SDZ 205 557 (n = 3) werc calculated.

4. Discussion

The current investigation has addressed the nature of second messenger coupling to the 5-HT 4 receptor in the tuniea muscularis mucosae of rat oesophagus. Acti-

vation of the 5-HT 4 receptor results in relaxation of smooth muscle (Baxter et al., 1991; Reeves et al., 1991), via a non-neuronal, postjunctional mechanism (Baxter et al., 1991; Gaynor et al., 1991). 5-HT 4 receptors present on mouse embryonic colliculi neurones and on membranes from guinea-pig hippocampus (Dumuis et al., 1988), as well as on cells in the atrial appendage of man (Kaumann et al., 1990), are positively coupled to adenylyl cyclase activity and increase the formation of cyclic AMP. However, the coupling preference of 5-HT 4 receptors in the rodent and guinea-pig digestive tract has not been established. Indeed, Linnik et al. (1991) failed to detect the involvement of adenylyl cyclase in 5-HT4-mediated responses in guinea-pig ileum. However, in this tissue, the neuronal location of the 5-HT 4 receptor (Craig and Clarke, 1990) may have led to dilution of the signal by nonneuronal tissue. The potencies of 5-HT and renzapride determined in the present study ( - l o g ECs0 values of 7.1 and 6.8) are similar to those reported for stimulation of cyclic AMP formation in mouse colliculi neurones by Dumuis et al. (1988, 1989), who also used a [3H]adenine incorporation assay (7.0 for 5-HT and 6.9 for renzapride). However, these values are somewhat lower than those obtained from whole tissue studies ( - l o g ECs0 values of 8.2 and 7.2, respectively; Baxter et al., 1991), indicating that the biochemical response to 5-HT 4 receptor activation is associated with lower effective receptor reserve (loss of post-receptor amplification). The equilibrium dissociation constant of a competitive antagonist for a receptor defines the receptor regardless of amplification processes or tissue location. In this regard the pA 2 estimate for tropisetron (6.7) made in the present study compares well with other estimates. These include relaxation of the rat oesophageal tunica muscularis mucosae (6.7; Baxter et al., 1991), stimulation of cyclic AMP production in mouse colliculi and guinea-pig hippocampus (6.2 and 6.3; Dumuis et al., 1988), contraction of the stimulated and quiescent guinea-pig ileum (6.4 and 6.3; Craig and Clarke, 1990; Eglen et al., 1990), contractile force of the human atrial appendage (6.7; Kaumann et al., 1991) and contraction of the guinea-pig ascending colon (6.4; Elswood et al., 1991). The recent introduction of SDZ 205 557 (Buchheit et al., 1991)provides an additional antagonist probe for characterisation of the 5-HT 4 receptor. Unlike tropisetron, which displays a much higher affinity for the 5-HT~ receptor, SDZ 205 557 offers true selectivity for the 5-HT 4 receptor with pA 2 estimates of between 7.1 and 7.5 (stimulated and quiescent guinea-pig ileum; Buchheit et al., 1991). SDZ 205 557 displayed an apparent pA 2 value of 7.7 in the present study, thus strengthening the definition of the receptor as a 5-HT 4 subtype.

12(I

In conclusion, stimulation of cyclic AMP formation by 5-HT in rat oesophageal tunica muscularis mucosae is mediated by the 5-HT 4 receptor. The pharmacological profile of the receptor matches closely that described for the rodent and guinea-pig brain and digestive tract, as well as the pig and human heart. The definition of the 5-HT 4 receptor is strengthened by 'fingerprinting' with SDZ 205 557, to date, the most selective antagonist known for the site. Finally, the present findings arc the first demonstration that 5-HT 4 receptor coupling in smooth muscle is mediated by cyclic AMP.

References Alvarez, R. and D.V. Daniels, 1990, A single column method for the assay of adenylatc cyclase. Anal. Biochem. 187, 98. Baxter, G.S., D.A. Craig and D.E. Clarke, 1991, 5-11ydroxytryptamine 4 receptors mediate relaxation of the rat oesophageal tunica muscularis mucosae, Naunyn-Schmicdeb. Arch. Pharmacol. 343, 439. Buchheit, K.H., R. Gamse and tl.-J. Pfannkuche, 1991, SDZ 21)5-557. a selective antagonist at 5-HT4 receptors in the isolated guinea-pig ileum, Eur. J. Pharmacol. 200, 373. Craig, D.A. and D.E. Clarke. 1990, Pharmacological characterisation of a neuronal receptor for 5-hydroxytryptamine in guinea-pig ileum with properties similar to the 5-hydroxytryptamine4 receptor, J. Pharmacol. Exp. Ther. 252. 1378.

Dumuis, A.. R. Bouhclal, M. Sebben, R. Cory and J. Bockaert, 1988, A non-classical 5-hydroxytryptamine receptor positively coupled with adenylatc cyclase in the central nervous system, Mol. Pharmacol. 34, 880. Dumuis, A., M. Scbben and J. Bockaert, 1989, BRL 24924: a potent agonist at a non-classical 5-HT receptor positively coupled with adenylate cyclase in colliculi neurons, Eur. J. Pharmacol. 162, 381. Eglen, R.M., S.R. Swank, L.K.M. Walsh and R.L. Whiting, 1990, Characterization of 5-HT3 and 'atypical' 5-HT receptors mediating guinea-pig ileal contractions in vitro, Br. J. Pharmacol. 101, 513. Elswood, C.J.. K.T. Bunce and P.P.A. Humphrey, 1991. Identification of putative 5-HT4 receptors in guinea-pig ascending colon, Eur. J. Pharmacol. 196, 149. Ford, A.P.D.W., R.M. Eglen, D.E. Clarke and G.S. Baxter, 1991, 5-Hydroxytryptamine (5-HT) stimulates adenylyl cyclase in rat oesophageal tunica muscularis mucosae (TMM) via non-classical 5-ttT receptors, Br. J. Pharmacol. 104, 46P. Gaynor, S.C., K.A. Wardle, B.F. King and G.J. Sanger, 1991, Location of 5-HT4 receptors in rat oesophagus. Br. J. Pharmacol. 104, 143P. Kaumann, A.J.. L. Sanders, A.M. Brown, K.J. Murray and M.J. Brown, 1990, A 5-hydroxytryptaminc receptor in human atrium, Br. J. Pharmacol. 100. 879. Linnik, M.D., B.T. Butler, L.A. Martin and N.K. Ahmed, 1991. Effect of benzamides on biochemical and motor responses at the 5-1IT4 receptor in the guinea-pig ileum, FASEB J. (Suppl. to Atlanta Meeting, April 1991), 5036P. Reeves, J.J., K.T. Buncc and P.P.A. llumphrcy. 1991, Investigation of the 5-hydroxytryptaminc receptor mediating smooth muscle relaxation in the rat oesophagus, Br. J. Pharmacol. 102, 1067.