Inhibitory effects of SR141716A on G-protein activation in rat brain

Abstract

N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride (SR141716A), a cannabinoid CB₁ receptor antagonist, has inverse agonist effects in cannabinoid CB₁ receptor-expressing cell lines, brain and peripheral organs. These studies characterized SR141716A-inhibited G-protein activity by measuring [³⁵S]GTPγS binding. Maximal inhibition of basal [³⁵S]GTPγS binding in cerebellar membranes was 50%. The EC₅₀ value for inhibition of [³⁵S]GTPγS binding was 4.4 μM, whereas the K_e for inhibition of R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazinyl]-(1-naphthalenyl)methanone mesylate (WIN 55,212-2)-stimulated [³⁵S]GTPγS binding was 0.6 nM. [³⁵S]GTPγS autoradiography was used to examine the regional specificity of SR141716A inhibition. SR141716A inhibited basal [³⁵S]GTPγS binding in all regions examined, with inhibition ranging from approximately 20% in caudate-putamen to 40% in hippocampus. These studies demonstrate that SR141716A is a competitive antagonist at nanomolar concentrations, whereas it inhibits basal receptor-mediated G-protein activity at micromolar concentrations. These data suggest that the apparent inverse agonist effect is either not cannabinoid CB₁ receptor-specific or that SR141716A is binding to different sites on the cannabinoid CB₁ receptor to produce inverse agonist versus competitive antagonist effects.

Introduction

Cannabinoid CB₁ receptors are the most numerous G-protein-coupled receptors in the brain, with levels tenfold higher than other G-protein-coupled receptors Devane et al., 1988, Herkenham et al., 1991. Although the role of the endogenous cannabinoid system has not yet been fully characterized, cannabinoid receptors are known to mediate the effects of Δ⁹-terahydrocannabinol (Devane et al., 1988), the active ingredient in marijuana (Gaoni and Mechoulam, 1964). Functional effects associated with cannabinoid receptor activation include antinociception, decreased spontaneous activity, hypothermia and impairment of short-term memory Dewey, 1986, Hollister, 1986. The characterization of cannabinoid receptors has been advanced by the development of selective and potent ligands, such as the cannabinoid CB₁ receptor-selective antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride (SR141716A) (Rinaldi-Carmona et al., 1994), which blocks cannabinoid CB₁ receptor agonist-mediated effects in both in vitro and in vivo preparations.

Many G-protein-coupled receptors exhibit spontaneous activity in the absence of agonist, which can be inhibited by inverse agonists Costa et al., 1990, Tian et al., 1994. The possibility that SR141716A acts as an inverse agonist at cannabinoid receptors was suggested by Compton et al. (1996) in studies showing stimulation of motor activity after administration of high doses (>3 mg/kg) of SR141716A. Subsequent studies using heterologous expression of cannabinoid CB₁ receptors in Chinese hamster ovary (CHO) cells demonstrated that SR141716A is an inverse agonist for cannabinoid-mediated effects on adenylyl cyclase and mitogen-activated protein kinase activity (Bouaboula et al., 1997). SR141716A inverse agonism has also been reported for in situ assays of brain (Gifford and Ashby, 1996) and isolated peripheral organs Pertwee and Fernando, 1996, Izzo et al., 1998, Coutts et al., 2000. In vivo administration of SR141716A also supports its role as an inverse agonist, although these data are complicated by the possibility that these effects are due to antagonism of endogenous cannabinoids Terranova et al., 1996, Richardson et al., 1997, Colombo et al., 1998a, Colombo et al., 1998b, Gessa et al., 1998, Rubino et al., 2000.

The inverse agonist effects of SR141716A in brain have been demonstrated using in vitro assays of G-protein and adenylyl cyclase activity (Meschler et al., 2000), as well as measures of ex vivo effector activity after in vivo administration of the drug (Rubino et al., 2000). The involvement of receptor-mediated G-protein activity in the inverse agonist response is supported by reports that SR141716A inhibits [³⁵S]GTPγS binding in cannabinoid CB₁ receptor-transfected cell lines Bouaboula et al., 1995, Landsman et al., 1997, MacLennan et al., 1998, neuronal cells and brain (Meschler et al., 2000). Data from cannabinoid CB₁ receptor-expressing CHO cells indicate that SR141716A inhibits effector responses that are mediated by G_iα (i.e. adenylyl cyclase) as well as Gβγ (i.e. mitogen-activated protein kinase) (Bouaboula et al., 1997). Moreover, studies in purified reconstituted systems have shown that SR141716A inhibits the activation of both G_i- and G_o-type G-proteins via spontaneously active cannabinoid CB₁ receptors (Glass and Northup, 1999). Ligand-modulated [³⁵S]GTPγS binding presents the opportunity to characterize SR141716A inverse agonism in brain because: (1) this methodology is used to investigate drug actions directly at the level of the G-protein and (2) moderate levels of basal [³⁵S]GTPγS binding in many regions indicate the presence of spontaneous receptor activity. Basal [³⁵S]GTPγS binding in autoradiographic assays exhibits a specific regional distribution (Sim et al., 1995), suggesting that specific receptor populations exhibit spontaneous activity. Furthermore, cannabinoid receptor signal transduction properties are known to vary by brain region Sim et al., 1995, Breivogel et al., 1997, Breivogel et al., 1999, but it is not clear whether this is also true for spontaneous activity and inverse agonist effects. These studies utilized ligand-modulated [³⁵S]GTPγS binding in rat brain membranes and sections to investigate the inverse agonist properties of SR141716A for G-protein activity.