- Top of page
- Conflict of interest
The neurotransmitter 5-hydroxytryptamine (5-HT) mediates a broad range of physiological and behavioural responses both centrally and peripherally (Saxena, 1995; Barnes and Sharp, 1999). All classes of the extensive 5-HT receptor family, except for the ligand-gated 5-HT3 receptor, are members of the seven transmembrane-spanning G protein-coupled family of receptors (Hoyer et al., 2002; Reeves and Lummis, 2002). These receptors modulate signal transduction pathways via stimulation or inhibition of adenylyl cyclase, modulation of cytosolic calcium concentration or activation of multiple alternative downstream effectors, including ERK1/2 (Pauwels, 2000; Norum et al., 2003).
The 5-HT4 receptor is thought to signal principally, but not exclusively, via Gs coupling to activation of adenylyl cyclase (Gerald et al., 1995; Pindon et al., 2002). Peripherally, activation of sinoatrial 5-HT4 receptors has a chronotropic effect on heart rhythm (Hegde and Eglen, 1996; Krobert et al., 2005; De Maeyer et al., 2006). In the digestive tract, activation of 5-HT4 receptors initiates a coordinated and complex prokinetic response that has been extensively studied and well described (Gershon, 1999). 5-HT4-mediated responses include modulation of cholinergic neurotransmission in the human proximal stomach (Leclere and Lefebvre, 2002) and increased colonic contractile responses in human, rat, dog and guinea-pig (Grider et al., 1998; Sakurai-Yamashita et al., 1999; Prins et al., 2000a, 2000b; Leclere et al., 2005). Contraction and potentiation of the electrical field twitch response in the guinea-pig ileum (Kajita et al., 2001) and colon (Jin et al., 1999) have also been described, as has facilitation of fast excitatory synaptic transmission (Galligan et al., 2003). In the rat oesophagus, 5-HT4 receptors mediate relaxation of smooth muscle (Leung et al., 1996; Goldhill et al., 1997).
Direct activation of 5-HT4 receptors has been demonstrated to relieve symptoms of several disease states, including functional bowel disorders, such as constipation-predominant irritable bowel syndrome and chronic constipation (Sanger, 1996; Alaradi and Barkin, 2002). Although the therapeutic potential of 5-HT4 agonists in the treatment of these disorders is accepted (Emmanuel et al., 2002; Lacy and Yu, 2002; Camilleri, 2004; Johanson, 2004), opportunities still exist for the development of novel agents with improved efficacy and/or tolerability profiles. Preclinical evaluations of novel 5-HT4 agonists include the use of human recombinant 5-HT4 receptor systems (Hinschberger et al., 2003). However, these systems have a number of limitations. Thus, questions surrounding the influence of receptor expression levels (Krobert et al., 2005), identity of relevant signal transduction pathways (Pindon et al., 2002) and desensitization mechanisms (Barthet et al., 2005), for example, prompt evaluation of novel agents in isolated tissue preparations, in particular from dog (Prins et al., 2000a), guinea-pig (Beattie et al., 2004) and/or human (Leclere et al., 2005) digestive tract. Guinea-pig tissue is readily accessible and guinea-pig pharmacological models are used routinely for the evaluation of 5-HT4 receptor selective agonists. Indeed, on the basis of work in guinea-pig isolated ileum, tegaserod has been described as a partial agonist (Buchheit et al., 1991). However, to date, although the guinea-pig 5-HT4 receptor has been cloned, there has been no comprehensive evaluation of the pharmacological properties of this recombinant receptor. The aims of the present study were to characterize the pharmacological properties of the guinea-pig recombinant 5-HT4 receptor in human embryonic kidney 293 (HEK293) cell stable transfectants, and compare these with native 5-HT4 receptors. A two-step approach was undertaken, in which binding affinities at the recombinant 5-HT4 receptor were compared with those at the native receptor in striatal membrane preparations, whereas functional comparisons were made with native guinea-pig 5-HT4 receptors, using the guinea-pig isolated colon longitudinal muscle myenteric plexus (LMMP) preparation. Although guinea-pig striatum has been used extensively to determine ligand-binding affinities (e.g. Grossman et al., 1993), to the best of our knowledge, there are no in vitro functional studies using guinea-pig striatum. In contrast, the use of guinea-pig colon preparations to profile 5-HT4 receptor ligands is well established (e.g. Leung et al., 1996). It should be noted, however, that there is an excellent correlation between the binding affinities for a range of agonist and antagonist ligands at 5-HT4 receptors in striatal and ileal/colon membrane preparations (Uchiyama-Tsuyuki et al., 1996). In addition, in the present study, the properties of the guinea-pig 5-HT4 receptor were compared directly with the prevalent human 5-HT4(b) receptor splice variant, with which it shares ∼95% identity, as well as high similarity within the C-terminus.
- Top of page
- Conflict of interest
The present study has demonstrated that the pharmacological properties of guinea-pig native and recombinant 5-HT4 receptors are very similar. Thus, there was a strong correlation between the binding affinities for a range of 5-HT4 receptor selective agonists and antagonists at 5-HT4 receptors in guinea-pig striatal membranes and the guinea-pig recombinant 5-HT4 receptor. Furthermore, the potency values of a panel of 5-HT4 receptor selective agonists for contraction of guinea-pig isolated colon LMMP preparations and in whole-cell cAMP accumulation assays, using HEK293 cells stably transfected with the recombinant receptor, were similar. The results of the present study confirm that 5-HT4 receptor density can influence the apparent potency and IA of 5-HT4 receptor agonists, as has been reported previously for other classes of G-protein-coupled receptor (Kenakin, 2002). These observations emphasize the importance of characterizing the functional activity of compounds of interest in recombinant systems expressing 5-HT4 receptors at densities that are believed to best approximate physiological levels. By extension, these studies also highlight the value of profiling at least subsets of compounds in relevant target tissues, whenever possible.
A wide variety of methodologies, ranging from functional analyses in isolated tissue preparations to immunohistochemical studies, have been used to describe the distribution of 5-HT4 receptors in mouse, rat, guinea-pig, dog and human digestive tract (Cohen et al., 1994; Sakurai-Yamashita et al., 1999; Prins et al., 2000a; Liu et al., 2005). In the present study, 5-HT4 mRNA transcripts were found ubiquitously in the guinea-pig digestive tract. The corresponding density of [3H]GR113808 binding sites in the ileum and proximal colon was ∼30 fmol mg−1 protein. This is consistent with values of 45 (Yoshikawa et al., 1998) or 12 (Uchiyama-Tsuyuki et al., 1996) fmol mg−1 protein, obtained previously for [3H]GR113808 binding to guinea-pig ileum. In humans, there has been no direct measurement of 5-HT4 receptor densities in the digestive tract. However, using autoradiographic techniques, 5-HT4 receptor binding sites have been identified in both the myenteric plexus and muscle layers of human colon (Sakurai-Yamashita et al., 1999). Interestingly, in the present study, 5-HT4 receptor mRNA transcripts were detected in guinea-pig oesophageal tissue, consistent with recent studies demonstrating 5-HT4 receptor immunoreactivity in the muscularis mucosae of the guinea-pig oesophagus (Poole et al., 2006).
The 10 5-HT4 receptor isoforms that have been identified in human tissues exhibit differential tissue distribution patterns (Medhurst et al., 2001; Vilaro et al., 2002; Brattelid et al., 2004) and the functional significance of these isoforms, which differ (with the exception of 5-HT4(h)) in the C terminal tail region is unclear. There is no evidence for an influence of C-terminal splice variant identity on 5-HT4 receptor binding affinity (e.g. Brattelid et al., 2004; Krobert et al., 2005). However, subtle differences in agonist potency and efficacy, receptor constitutive activity, signal transduction pathways and receptor desensitization properties have been reported for the different splice variants (Claeysen et al., 1999; Bender et al., 2000; Pindon et al., 2002; Mialet et al., 2003). For the guinea-pig, although a sequence encoding a single 5-HT4 receptor variant was submitted to Genebank in 1997, to the best of our knowledge, there has been no publication describing the recombinant receptor. The present study has focused on this variant, which has close amino-acid sequence similarity to the human 5-HT4(b) receptor splice variant and probably represents the guinea-pig orthologue of this receptor. It is hypothesized that in the guinea-pig this is the most abundant form of the receptor. However, a more thorough cloning strategy using primers encoding the specific C-terminal of each receptor variant would be required to definitively rule out the presence of alternative variants.
The pharmacological and functional properties of the guinea-pig recombinant 5-HT4 receptor, in general, were very similar to those of guinea-pig native receptors. As expected, the pKi values for a range of 5-HT4 receptor selective agonists and antagonists at the recombinant receptor were similar to those at native 5-HT4 receptors in guinea-pig striatal (present study; Uchiyama-Tsuyuki et al., 1996) and ileal (Uchiyama-Tsuyuki et al., 1996) membrane preparations.
The functional properties of guinea-pig recombinant 5-HT4 receptors were characterized using whole-cell cAMP accumulation studies on two different clones: (i) gp 5-HT4 – clone 2, for which the 5-HT4 receptor density (∼3 pmol mg−1 protein) was of a similar magnitude to that reported for several clones that have been used in the characterization of human 5-HT4 receptor splice variants (Bach et al., 2001; Pindon et al., 2002; Krobert et al., 2005) and (ii) gp 5-HT4 – clone 1, for which the 5-HT4 receptor density was at least 10-fold lower (∼0.2 pmol mg−1 protein) and proposed to be closer to physiological levels. Thus, although the 5-HT4 receptor density in gp 5-HT4 – clone 1 is 5- to 10-fold higher than that in guinea-pig isolated ileum or proximal colon, it is very similar to that in guinea-pig striatal membranes.
The rank order of 5-HT4 receptor agonist potency, tegaserod >5-HT=prucalopride >cisapride >metoclopramide=mosapride, was similar for the lower receptor density clone and the guinea-pig isolated colon LMMP preparation (present study; Mine et al., 1997). Tegaserod was a full agonist (relative to 5-HT) at the guinea-pig recombinant receptor, in contrast to prucalopride, and the benzamide analogues, cisapride and mosapride, which were partial agonists (IA values=22–76%). Cisapride and mosapride are also 5-HT4 receptor partial agonists (IA values=37–74%) in isolated tissue preparations from several species including guinea-pig (present study; Bach et al., 2001). In the present study, the potency and IA of mosapride (pEC50 <5.4 and IA <37%, respectively) were similar at both the recombinant guinea-pig 5-HT4 receptor and in the guinea-pig LMMP preparation. The present findings suggest that receptor density significantly influences the functional properties of a range of 5-HT4 agonists at the guinea-pig 5-HT4 receptor. There was a significant (5- to 10-fold) increase in agonist potency in the higher receptor density cell line, gp 5-HT4 – clone 2, relative to that in gp 5-HT4 – clone 1, although the rank order of agonist potency was unchanged. In addition, all the agonists tested were full agonists (relative to 5-HT) in gp 5-HT4– clone 2. Therefore, consistent with previous observations at the human 5-HT4(a) and 5-HT4(b) receptors (Pindon et al., 2002), the partial agonist cisapride behaved as full agonist in the higher receptor density clone.
Several authors have noted a similar dependence of 5-HT4 agonist potencies and/or IA values on human 5-HT4 receptor density (Bach et al., 2001; Krobert et al., 2005). For example, a receptor density-dependent increase in agonist efficacy is thought to underlie the different IA values for prucalopride at human 5-HT(4a, b, c and i) receptor splice variants (Krobert et al., 2005). These observations are consistent with the operational model of agonism (Black and Leff, 1983). Thus, at a higher density of transfected receptors there is amplification of the receptor-induced signals, that is spare receptors (Bruheim et al., 2003).
It is well established that tegaserod is a potent, partial agonist at 5-HT4 receptors in rodent isolated tissue preparations (Buchheit et al., 1991; Beattie et al., 2004). In contrast, tegaserod was a full agonist at the recombinant guinea-pig 5-HT4 receptor, when expressed at either low or high receptor density. Tegaserod is also a potent, full agonist at human 5-HT4(a) and 5-HT4(b) receptor splice variants (Pindon et al., 2002). Further investigation is warranted to understand the apparent discrepancies between the isolated tissue and recombinant data. Recently, De Maeyer et al. (2006), using the operational model of agonism, demonstrated that the efficacy of tegaserod is in fact similar (equivalent τ-values) to that of 5-HT in porcine atrium and stomach in vitro preparations. The potency of tegaserod, however, was lower than expected and the authors attributed this to poor tissue penetration and/or partial precipitation, because of low solubility. Other factors which also may influence the tegaserod response in the recombinant and isolated tissue preparations are differential receptor binding kinetics, receptor reserve, splice variant identity, stimulus-response coupling efficiency, activation of alternate signalling pathways and/or receptor desensitization. However, such factors might be expected to influence the response to other 5-HT4 receptor agonists unless phenomena like agonist-directed trafficking of the stimulus response occurs. To date, there is no evidence that agonist-directed trafficking of 5-HT4 receptors, as has been previously described for the 5-HT2(a) and 5-HT2(c) receptors (Berg et al., 1998), occurs. Alternatively, tegaserod may interact with non-5-HT4 receptors in the tissue preparations, thereby influencing the magnitude of 5-HT4 receptor-mediated responses. However, the contribution of 5-HT4 receptors to the tegaserod-evoked contractile responses in digestive tract tissues has been confirmed using 5-HT4 receptor selective antagonists such as piboserod (present study; Beattie et al., 2004). Collectively, these data suggest that tegaserod is a full agonist at 5-HT4 recombinant receptors and additional factors underlie its apparent poor efficacy in tissue preparations.
Finally, the present study has demonstrated that the pharmacological and functional properties of the guinea-pig recombinant 5-HT4 receptor are very comparable to those of human recombinant 5-HT4(b) receptor splice variants (Mialet et al., 2000; Pindon et al., 2002). Given the potential influence of receptor density on the functional properties of human (Bach et al., 2001; Bruheim et al., 2003; Krobert et al., 2005) and/or guinea-pig (present study) 5-HT4 receptors, two clones that expressed the respective receptors at a similar density (Bmax=0.21±0.03 and 0.34±0.03 pmol mg−1 protein, respectively) were selected for the comparison in the present study. There was a strong correlation between the agonist potency values for the human and guinea-pig recombinant receptors and very good agreement between the corresponding IA values, with the exception of prucalopride and cisapride, which behaved as full agonists in the human 5-HT4(b) recombinant cell line. A number of investigators have evaluated the pharmacological properties of 5-HT4 receptors in human isolated tissue preparations (Tam et al., 1995; Prins et al., 2000b; Leclere et al., 2005). In spite of the significant limitations associated with use of such tissue preparations there was a reasonable correlation with the guinea-pig recombinant data.
In conclusion, the present study has demonstrated that ligand binding affinities and functional properties at the guinea-pig recombinant 5-HT4 receptor are similar to those at guinea-pig native receptors, in striatal and colonic tissue, respectively. Furthermore, the guinea-pig recombinant 5-HT4 receptor and human 5-HT4(b) receptor splice variant share very similar pharmacological properties. Collectively, these data suggest that pharmacological models using the guinea-pig recombinant 5-HT4 receptor and/or isolated colon preparations represent relevant, and important, tools for the identification and/or characterization of novel 5-HT4 receptor agonists. Such agents may offer improved efficacy for the treatment of disorders of reduced gastrointestinal motility. The pharmacological rationale for the different agonist profiles of tegaserod and the extent to which this may influence the clinical efficacy of tegaserod as a prokinetic agent require elucidation.