The Action of SDZ 205557 at 5-Hydroxytryptamine
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Br. J. Pharmacol. (1993), 108, 376-382 19" Macmiflan Press Ltd, 1993 The action of SDZ 205,557 at 5-hydroxytryptamine (5-HT3 and 5-HT4) receptors 1R.M. Eglen, R. Alvarez, L.G. Johnson, E. Leung & E.H.F. Wong Institute of Pharmacology, Syntex Discovery Research, 3401 Hillview Ave, Palo Alto, CA 94304, U.S.A. 1 The interaction of the novel antagonist, SDZ 205,557 (2-methoxy-4-amino-5-chloro benzoic acid 2-(diethylamino) ethyl ester), at 5-HT3 and 5-HT4 receptors has been assessed in vitro and in vivo. 2 In guinea-pig hippocampus and in the presence of 0.4 JLM 5-carboxamidotryptamine, 5-HT4-mediated stimulation of adenylyl cyclase was competitively antagonized by SDZ 205,557, with a pA2 value of 7.5, and a Schild slope of 0.81. In rat carbachol-contracted oesophagus, 5-HT4-receptor mediated relaxations were surmountably antagonized by SDZ 205,557 with a similar pA2 value (7.3). This value was agonist-independent with the exception of (R)-zacopride, against which a significantly lower value (6.4) was observed. 3 In functional studies of 5-HT3 receptors, SDZ 205,557 exhibited an affinity of 6.2 in guinea-pig ileum compared with 6.9 at binding sites labelled by [3H]-quipazine in NG108-15 cells. In the anaesthetized, vagotomized micropig, SDZ 205,557 produced only a transient blockade of 5-HT4-mediated tachycardia. This contrasted with tropisetron, which was active for over 60 min after administration. The half-lives for the inhibitory responses of SDZ 205,557 and tropisetron were 23 and 116 min, respectively. 4 In conclusion, SDZ 205,557 has similar affinity for 5-HT3 and 5-HT4 receptors. The apparent selectivity observed in guinea-pig is due to the atypical nature of the 5-HT3 receptor in this species. The short duration of action of this novel antagonist may complicate its use in vivo. SDZ 205,557 should, therefore, be used with appropriate caution in studies defining the 5-HT4 receptor. Keywords: SDZ 205,557; 5-HT3 receptors; 5-HT4 receptors Introduction The 5-hydroxytryptamine (5-HT) receptor family may be notably, exhibits lower affinities for ligands than either rat or classified into 5-HTI, 5-HT2, 5-HT3 (Bradley et al., 1986) and mouse 5-HT3 receptors (see Kilpatrick & Tyers, 1992, for 5-HT4 subgroups (Bockaert et al., 1992). The 5-HT4 receptor review). This may lead to an overestimation of the selectivity (Dumuis et al., 1988) is stimulated by indoles (such as 5-HT, of antagonists, including SDZ 205,557, in favour of the 5-methoxytryptamine, 5-carboxamidotryptamine), substituted 5-HT4 receptor (Bockaert et al., 1992). In the present study, benzamides (such as renzapride, zacopride, cisapride) and SDZ 205,557 has been further evaluated by use of in vitro azabicycloalkyl benzimidazolones (such as BIMU-1, BIMU- and in vivo responses in various species, on preparations 8), but is insensitive to antagonists of 5-HT1, 5-HT2, and sensitive to 5-HT4 stimulation. This receptor, in contrast to 5-HT3 receptors, such as methysergide, ketanserin and the 5-HT3 receptor, appears to exhibit affinities that are not ondansetron, respectively (see Craig & Clarke, 1990; species-dependent (Bockaert et al., 1992). Bockaert et al., 1992 for reviews). The 5-HT4 receptor is A preliminary account of these studies has been previously competitively antagonized by tropisetron (ICS 205,930; - log published (Eglen et al., 1992). KB= 6.5) and DAU 6285 (-log KB = 6.8; Dumuis et al., 1992). Receptors exhibiting this pharmacological profile have been demonstrated in embryonic mouse colliculi neuronal Methods cultures (Dumuis et al., 1992), guinea-pig ileum (Craig & Clarke, 1990; Eglen et al., 1990) and colon (Elswood et al., Guinea-pig hippocampal adenylyl cyclase 1991), rat oesophagus (Baxter et al., 1991; Waikar et al., 1992), porcine myocardium (Kaumann, 1990), human myo- Male guinea-pigs (Hartley, 250-500 g) were killed by CO2 cardium (Kaumann et al., 1990) and amphibian adrenal asphyxiation. The hippocampi were then rapidly dissected, gland (Idres et al., 1991). More precise definition of 5-HT4 and homogenized (20 strokes) in a glass homogenizer in 9 ml receptors is, however, hampered by the lack of selective, of ice-cold buffer, of the following composition (mM): sucrose competitive antagonists (see Turconi et al., 1991; Bockaert et 300, Tris-HCl 20, EGTA 1, Na2EDTA 2.5 and dithiothreitol al., 1992 for reviews). 1 (pH 7.4, 23°C). This homogenate was then diluted, 1:8 Buchheit et al. (1991) reported that SDZ 205,557 (2- (v/v) with buffer and centrifuged (10 min, 39,000 g, 4C). The methoxy4-amino-5-chloro benzoic acid 2-(diethylamino) ethyl pellet was resuspended in 9 ml of buffer and the membrane ester) selectively antagonized 5-HT4 receptors in guinea-pig suspension used directly in the adenylyl cyclase assay. isolated ileum with an affinity (- log KB) of 7.5. This affinity Antagonists were added 30 min prior to addition of agonist. was greater than that at 5-HT3 receptors in this tissue (- log Measurements of adenylyl cyclase activity were performed, KB = 6.0). SDZ 205,557 lacked affinity at 5-HTI or 5-HT2 three times, according to the method of Alvarez & Daniels receptors and was, therefore, suggested as a useful ligand to (1990) with each sample run in triplicate. The final composi- define the 5-HT4 receptor (Buchheit et al., 1992). Phar- tion of the incubation medium was (mM): [at32P]-adenosine macological evidence suggests that 5-HT3 receptors are a 5'-triphosphate ([&32P]-ATP, 0.25 pCi) 0.5, MgSO4 5, Tris- family of species variants. The guinea-pig 5-HT3 receptor, HCl 44 (pH 7.4), 1-methyl-3-isobutylxanthine 1.0, sucrose 50, EDTA 1.0, EGTA 0.2, dithiothreitol 0.2, adenosine 3':5'- cyclic monophosphate (cyclic AMP) 2, guanosine 5'-triphos- I Author for correspondence. phate (GTP) 0.1, phosphoenol pyruvate 20 and pyruvate ACTION OF SDZ 205,557 AT 5-HT3 AND 5-HT4 RECEPTORS 377 kinase 6 units ml-'. The reaction was initiated by the sequen- 205,557 were used, and the slope of the Schild plot deter- tial addition of the membrane suspension (40 #Al) to the mined by regression analysis. incubation medium in a total reaction volume of 200 Jl. Incubations were performed for 30 min at 370C and ter- Competition radioligand binding studies minated by the addition of 20 ftl of a stock solution of [3H]-cyclic AMP (0.005 JCi) in 2.2 N HCl. Labelled cyclic Membranes were prepared from NG108-15 cells cultured AMP was added to estimate and correct for recovery of the under conditions described previously (Sharif et al., 1991). In nucleotide following column chromatography. The tube con- competition binding studies, 5-HT3 receptors in NG108-15 tents were heated at 95C for 4 min and then cooled in an cell membranes were labelled with 0.5 nM [3H]-quipazine and ice-water bath. Neutral alumina (1.3 g) was dispensed into non-specific binding was defined by 1 JAM (S)-zacopride. The disposable polypropylene columns with a Uniflow adjustable membrane homogenates were incubated in 25 mM Tris-Krebs powder measure. The columns were placed on a plexiglas (pH 7.4 at 250C) with radioligand and varying concentrations rack designed to hold the columns and to fit over a box of of SDZ 205,557, or other standard 5-HT3 receptor anta- 100 scintillation vials. An aliquot (200 pl) of the solution gonists, in a final assay volume of 0.5 ml. The incubations contained in each tube was pipetted onto the columns and were carried out for 45 min at 250C and were terminated by allowed to flow into the dry alumina. Cyclic AMP was then rapid vacuum filtration over Whatman GF/B filters using a eluted with 4 ml of 0.1 M ammonium acetate, pH 7. The Brandell 48 cell harvester. This was immediately followed by effluent (3.2 ml) was collected into scintillation vials, mixed 8 s of washing with ice-cold 0.1 M NaCl. The filters were with 15 ml scintillation fluid and counted for 3H and 32p in a pretreated with 0.3% polyethyleneimine in order to reduce liquid scintillation spectrometer. filter binding of the radioligand and the radioactivity retained on the filters was determined by liquid scintillation spect- rometry. All competition data were analyzed by iterative Rat isolated oesophageal muscularis mucosae curve fitting procedure as described previously (Michel & Whiting, 1984). The apparent dissociation constant (K.) of The method used was that previously described by Baxter et competing ligands was calculated from ICso values by the al. (1991). Male rats (Sprague-Dawley, 200-250 g) from Cheng-Prusoff equation (Cheng & Prusoff, 1973). Charles River were killed by CO2 asphyxiation, the thoracic oesophagus removed and placed in Tyrode solution (com- Anaesthetized micropig studies position, mM: NaCl 139.0, KCl 2.7, MgCl2 6H2O 1.1, NaH2PO4 0.4, glucose 5.6, NaHCO3 11.8 and CaCl2-6H2O The method used was modified from that described by Villa- 1.8). The outer proprial muscle coat was cut longitudinally lon et al. (1990). Yucatan micropigs (male and female; and gently peeled away, leaving the inner muscularis 14.9-20.8 kg, Charles River Laboratories Inc., Wilmington, mucosae. Silk threads were then tied through the lumen on MA, U.S.A.) were pretreated with ketamine HCl (approx. both ends of the tissues, which were then mounted vertically 30 mg kg-', i.m.), anaesthetized with pentobarbitone sodium in 10 ml tissue baths containing Tyrode solution with 1 JAM (20 mg kg-') via a marginal ear vein, intubated, and methysergide, 30 gM cocaine and 30 JM corticosterone.