The putative «silent» 5-HT1A receptor antagonist, WAY 100635, has inverse agonist properties at cloned human 5-HT1A receptors
Introduction
A generally accepted interpretation of the cycle of events by which G protein-coupled receptors elicit responses is that agonist binding to G protein-coupled receptors is required to induce the formation of a receptor–G protein complex, and subsequent guanosine 5′-diphosphate/guanosine 5′-triphosphate (GDP/GTP) exchange. In the absence of the agonist, the receptor is thought to exist entirely in the dissociated state, corresponding to the lower affinity component of biphasic agonist displacement in radioligand binding assays. The ternary complex, consisting of the agonist, receptor, and G protein, accounts for the higher affinity component in such assays. It has become apparent in the last few years that while this description is reasonable for receptors coupled primarily or exclusively to Gs (such as the β2-adrenoceptor), receptors that couple primarily with pertussis toxin-sensitive G proteins (Gi or Go), and show Na+-dependent agonist and antagonist binding affinities may behave quite differently. These latter receptors (e.g., δ-opioid receptor, α2-adrenoceptor, 5-HT1Dα/1Dβ) appear to form receptor–G protein complexes and promote GDP/GTP exchange even when not occupied by agonists, and do so in a manner that can be affected by monovalent cations (most notably Na+) Costa et al., 1990, Jagadeesh et al., 1990, Tian and Deth, 1993, Gray et al., 1997.
Inverse agonism has been described in the in vitro experimental models such as transfected cell lines, in which the receptors are present in sufficiently high densities to cause a significant activation of the signal transduction systems for the inverse agonist to inhibit. For instance, inverse agonist activity of spiperone, but not of N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]-N-(2-pyridinyl)cyclohexanecarboxamide (WAY 100635), has been demonstrated using guanosine 5′-0-(3-[35S]thio)-triphosphate binding ([35S]GTPγS binding) to membranes from Chinese hamster ovary (CHO) cells expressing 1.6 pmol/mg protein of human recombinant 5-hydroxytryptamine1A (5-HT1A) receptors (Newman-Tancredi et al., 1997).
Inverse agonism can be affected by variations in [Na+]. It has been reported that α2-adrenergic receptor antagonists decreased [35S]GTPγS binding to membranes from PC-12 cells, stably expressing the α2D-adrenoceptors, and that their inverse agonist activity was decreased by increasing the NaCl concentration (Tian et al., 1994). In contrast, agonists' responses were increased at higher NaCl levels (Tian et al., 1994). [35S]GTPγS binding to membranes from human epithelioid carcinoma (HeLa) cells expressing cloned human 5-HT1A receptors (500 fmol/mg protein) (HA7; Fargin et al., 1989), in the presence of 100 mM NaCl, has been used previously to examine the pharmacological properties of 5-HT1A receptor ligands (Stanton and Beer, 1997). Here, we investigated the effects of varying the Na+ concentrations on the activity of 5-HT1A receptor ligands, such as WAY 100635, to alter [35S]GTPγS binding to membranes prepared from HA7 cells. Part of the present results has been reported previously in abstract form at the Fourth IUPHAR Satellite Meeting on Serotonin in 1998.
Section snippets
Cell culture
HA7 cells were grown in Dulbecco modified Eagle medium (DMEM) (GIBCO) supplemented with 10% foetal calf serum, gentamicin (100 μg/ml) and geneticin (G418) (400 μg/ml), in 5% CO2 at 37°C in a water-saturated atmosphere. The cells were plated in 150 cm2 petri dishes until they reached a 90–100% confluence, after which, they were washed with phosphate buffered saline (PBS) and stored at −80°C. In some experiments, the cells were treated with pertussis toxin (200 ng/ml) for 16 h, then washed with
Potency and intrinsic activity of serotonergic compounds in standard (100 mM) NaCl conditions
The ability of a variety of compounds with affinity for 5-HT1A receptors to affect basal [35S]GTPγS binding to membranes from HA7 cells was investigated using a reaction buffer that included 100 mM NaCl, as described previously (Stanton and Bear, 1997). Under these conditions, positive intrinsic activity (EMAX) ranged from 419.4±11.70% of basal [35S]GTPγS binding for (+)-8-OH-DPAT to 117.6±4.51% for WAY 100135 (Table 1). Serotonin stimulated basal [35S]GTPγS binding with a pEC50 of 7.31±0.036
Discussion
The effects of a variety of 5-HT1A receptor ligands on basal [35S]GTPγS binding to membranes from HA7 cells were investigated, using the binding conditions previously described by Stanton and Bear (1997) with 100 mM NaCl. The intrinsic activity and pEC50 values obtained here correlated with those reported by Newman-Tancredi et al. (1998), who used [35S]GTPγS binding to membranes from CHO cells expressing human recombinant 5-HT1A receptors.
p-MPPI and spiperone, described as putative inverse
Acknowledgements
The authors thank Nathalie Leduc for her excellent technical assistance.
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