Constitutive Activity of 5HT2C Receptors In vitro Consideration and Pharmacology

The constitutive activity of seven-transmembrane receptor is a concept now widely accepted and well characterized in heterologous recombinant systems in vitro (Costa and Herz 1989; Milligan et al. 1995; Berg et al. 2001; Kenakin 2001, 2004). The capacity of this receptor to regulate cellular signaling systems in the absence of occupancy by a ligand could be considered as an artificial property as it depends on the density of the receptor at cell surface and the total absence of the endogenous ligand, two conditions that can be easily controlled in vitro (Milligan et al. 1995; Milligan and Bond 1997). Nevertheless, a large number of seven-transmembrane receptors exhibit constitutive activity in vitro (Seifert and Wenzel-Seifert 2002), which suggests that inverse agonism might have broad pharmacological relevance.

5-HT2C receptors couple to multiple effectors systems (Fig. 10.1) (Berg et al. 2008) and multistate receptor models predict that constitutive activity should depend on the responses measured (De Deurwaerdere et al. 2004; Berg et al. 2005). It has been shown that 5-HT2C receptors display a higher degree of constitutive activity towards phospholipase C (PLC) compared with phospholipase A2 (PLA2) (Barker et al. 1994; Berg et al. 1999; Herrick-Davis et al. 1999; Niswender et al. 1999). Numerous 5-HT2C compounds previously thought to act as antagonists, such as mesulergine, ritanserin, mianserin, and the prototypical atypical antipsychotic drugs clozapine and olanzapine, have been characterized as inverse agonists in vitro on PLC signaling pathway (Fig. 10.1b) (Rauser et al. 2001). Indeed, these compounds decrease the high basal response induced by constitutively active 5-HT2C receptors on myo-[3H]inositol phosphate (IP) accumulation (Berg et al. 1998, 1999, 2005; Barker et al. 1994). Their effect is opposite to the effects of 5-HT2C agonists in these heterologous systems. Further, 5-HT2C antagonists are able to block, in a concentration-dependent manner, the effects induced by both agonists and inverse agonists (Fig. 10.1a) (Berg et al. 1998; Kennett et al. 1996, 1997; Price et al. 2001). In Chinese hamster ovary (CHO) cells expressing human 5-HT2C receptors at a density to optimize its constitutive activity (Berg et al. 1998), SB 206553 behaves as a strong inverse agonist at PLC-dependent, PLA2-dependent, and activation of Ga-dependent responses (De Deurwaerdere et al. 2004). SB 242084 is equally

Fig. 10.1 Pharmacological properties of 5-HT2C inverse agonists (SB 206553 and clozapine) and 5-HT2C antagonists (SB 242084 and SB 243213) in CHO-1C7 cells. The effects of each compound were measured as the percentage of basal activity of three effectors systems: PLC, PLA2 and activation of Gai. Respectively, the amount of myo-[3H]inositol phosphate (IP) accumulated, [3H] arachidonic acid (AA) released, and [35S]GTPgS bound was determined after 25 min of incubation with the indicated drugs. (a) Basal IP accumulation, AA release, and [35S]GTPgS binding levels were as follows: 5,378 ± 789 dpm, 1,544 ± 167 dpm, and 72 ± 13 pmol/mg protein, respectively. Data are expressed as the mean ± SEM of four to six independent experiments. (b) Basal IP accumulation, AA release, and [35S]GTPgS binding levels were as follows: 6,577 ± 407 dpm, 1,222 ± 42 dpm, and 89 ± 7 pmol/mg protein, respectively. Data are expressed as the mean ± SEM of three to five independent experiments (*p < 0.05 compared with the effect of SB 206553, Student's paired t test) (a: From De Deurwaerdere et al. 2004. Copyright permission from the Society for Neuroscience. b: Adapted from Berg et al. 2006. Copyright permission from the American Society for Pharmacology and Experimental Therapeutics)

Fig. 10.1 Pharmacological properties of 5-HT2C inverse agonists (SB 206553 and clozapine) and 5-HT2C antagonists (SB 242084 and SB 243213) in CHO-1C7 cells. The effects of each compound were measured as the percentage of basal activity of three effectors systems: PLC, PLA2 and activation of Gai. Respectively, the amount of myo-[3H]inositol phosphate (IP) accumulated, [3H] arachidonic acid (AA) released, and [35S]GTPgS bound was determined after 25 min of incubation with the indicated drugs. (a) Basal IP accumulation, AA release, and [35S]GTPgS binding levels were as follows: 5,378 ± 789 dpm, 1,544 ± 167 dpm, and 72 ± 13 pmol/mg protein, respectively. Data are expressed as the mean ± SEM of four to six independent experiments. (b) Basal IP accumulation, AA release, and [35S]GTPgS binding levels were as follows: 6,577 ± 407 dpm, 1,222 ± 42 dpm, and 89 ± 7 pmol/mg protein, respectively. Data are expressed as the mean ± SEM of three to five independent experiments (*p < 0.05 compared with the effect of SB 206553, Student's paired t test) (a: From De Deurwaerdere et al. 2004. Copyright permission from the Society for Neuroscience. b: Adapted from Berg et al. 2006. Copyright permission from the American Society for Pharmacology and Experimental Therapeutics)

effective as an inverse agonist towards PLA2 and Gai activation but displays low-efficacy agonism toward PLC (De Deurwaerdere et al. 2004). SB 243213 behaves as a partial inverse agonist at PLA2 and as a full inverse agonist at Gai activation, while it displays neutral antagonism at PLC (Burns et al. 1997). SB 243213 and SB 242084 correspond pharmacologically to protean ligands (Kenakin 2001; Berg et al. 2005). According to their pharmacological properties at PLC-dependent responses, SB 242084 and SB 243213 induce a rightward shift of the inhibition of IP accumulation induced by SB 206553 (Fig. 10.1a) (De Deurwaerdere et al. 2004; Berg et al. 2006).

Other cellular effectors such as desensitization mechanisms occurring via G-protein-coupled receptor kinase (GRK) and receptor internalization (down-regulation) are targeted by the constitutive activity of 5-HT2C receptors (Berg et al. 1999, 2006). These mechanisms triggered by a prolonged exposure of the receptor to inverse agonists lead to enhanced responsiveness of both ligand-dependent and ligand-independent 5-HT2C receptor activation through the G -PLC signaling pathway without change in receptor density (Berg et al. 1999). The molecular plasticity of the 5-HT2C receptor is further supported by the posttran-scriptional process of editing. Adenosine-to-inosine editing events of the 5-HT2C mRNA leads to changes in amino acids in the second intracellular loop of the receptor, a domain known to play an important role in G-protein coupling, desen-sitization mechanisms, and isomerization. All these mechanisms are able to modulate the level of constitutive receptor activity (Burstein et al. 1995; Marion et al. 2004; Werry et al. 2008). RNA editing generates different mRNA transcripts that encode for numerous receptor isoforms (Burns et al. 1997; Fitzgerald et al. 1999). In general, the constitutive activity of RNA-edited isoforms of the 5-HT2C receptor towards PLC is reduced compared with the nonedited receptor (Berg et al. 2005, 2008; Herrick-Davis et al. 1999; Niswender et al. 1999). Along with a specific distribution of RNA-edited 5-HT2C isoforms throughout the brain (Burns et al. 1997) and a regulation by 5-HT (Gurevich et al. 2002; Schmauss 2005), it has been suggested that this process may allow for a fine-tuning of 5-HT transmission via the 5-HT2C receptor (Berg et al. 2008; Herrick-Davis et al. 1999; Niswender et al. 1999).

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