Dual intracellular signaling pathways mediated by the human cannabinoid CB1 receptor
Introduction
Interest in cannabinoid research has intensified in recent years following the cloning of specific cannabinoid receptor subtypes: the cannabinoid CB1 receptor from rat (Matsuda et al., 1990), human (Gérard et al., 1991) and mouse (Chakrabarti et al., 1995) brain, and the cannabinoid CB2 receptor from human (Munro et al., 1993) and mouse (Shire et al., 1996b) peripheral tissues, and following the discovery of endogenous ligands (Devane et al., 1992; Mechoulam et al., 1995) and the highly-specific cannabinoid CB1 and CB2 receptor antagonists SR 141716A {N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride} (Rinaldi-Carmona et al., 1994) and SR 144528 {N-[(1S)-endo-1,3,3-trimethyl bicyclo [2.2.1]heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methoxybenzyl)-pyrazole-3-carboxamide} (Rinaldi-Carmona et al., 1998), respectively. Both the central cannabinoid CB1 and peripheral cannabinoid CB2 receptors are members of the heptahelical G protein-coupled receptor superfamily. The cannabinoid CB2 receptor is known to mediate its effects through the pertussin toxin-sensitive Gi/o inhibition of adenylyl cyclase (Bayewitch et al., 1995). The cannabinoid CB2 receptor can also mediate βγ-mediated intermediate-early gene expression through the mitogen-activated protein (MAP) kinase pathway and this is also blocked by pertussis toxin treatment (Bouaboula et al., 1996). The cannabinoid CB1 receptor, apart from exhibiting all the same functions as the cannabinoid CB2 receptor, also inhibits N- and Q-type voltage-dependent Ca2+ channels and stimulates inwardly rectifying K+ currents (see Pertwee, 1997for a recent review). In the past, a functional link with the inositol phosphate/Ca2+ pathway could not be established despite numerous investigations with diverse tissues containing cannabinoid CB1 receptors (Howlett, 1995). Recently, however, a rapid, transient elevation of intracellular free Ca2+ upon agonist binding to cannabinoid CB1 receptors in neuroblastoma×glioma NG108-15 and NG18TG2 cells, mediated by pertussis toxin-sensitive G proteins, has been observed (Sugiura et al., 1996, Sugiura et al., 1997, Sugiura et al., 1999).
In addition to the multiple signaling functions implicating the cannabinoid CB1 receptor, there were several reports prior to the cloning and characterization of the receptor that suggested that under certain conditions cannabinoids could also promote the accumulation of cAMP in cell cultures and tissue homogenates [reviewed in (Pertwee, 1988)]. More recently, it was shown (Glass and Felder, 1997; Felder et al., 1998) that pertussis toxin treatment of the cannabinoid CB1 receptor attenuated agonist-mediated inhibition of adenylyl cyclase and unmasked an agonist-mediated stimulation of the enzyme. However, the stimulation necessitated the action of a costimulant such as forskolin before the effect could be seen. It was reported (Maneuf and Brotchie, 1997) that high concentrations of the potent cannabinoid WIN 55,212-2 {((R)-(+)[2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl](1-naphthalenyl)methanone}, with an EC50 of 32.8 μM, could stimulate basal cAMP accumulation in a slice preparation of globus pallidus in the absence of either costimulant or pertussis toxin and that the effects could be blocked by the antagonist SR 141716A, thereby clearly showing that they were mediated by the cannabinoid CB1 receptor. More recently, the potency and intrinsic activity of various cannabinoid receptor ligands in stimulating or inhibiting cAMP accumulation were quantified (Bonhaus et al., 1998).
We are at present investigating structural features of the cannabinoid CB1 receptor implicated in signal transduction using chimeric CB1/CB2 and mutated wild type cannabinoid receptors. To study cannabinoid CB1 receptor modifications that may have repercussions on downstream gene transcription, in addition to direct cAMP assays, we are making use of a reporter gene system consisting of a minimal promoter containing cAMP-response elements (CRE) fused to the firefly luciferase coding region (Bouaboula et al., 1997). We discovered that CP 55,940 could induce cAMP accumulation in both a Chinese hamster ovary (CHO) cell line, CHO-CB1, and in CHO cells transiently expressing the receptor. We subsequently cotransfected CHO cells with expression vectors for the human cannabinoid CB1 receptor and the CRE-luciferase fusion and discovered that the basal luciferase activity increased following treatment of the cells with the cannabimimetic CP 55,940 {(−)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl) cyclohexan-1-ol} in a strictly dose–response manner, or after incubation with pertussis toxin, the two effects being additive. SR 141716A counteracted the effect of the agonist, also in a dose–response manner, showing that the luciferase induction was cannabinoid CB1 receptor-mediated. Since the cannabinoid CB2 receptor failed to produce a response after coexpression with the CRE-luciferase cassette, using chimeric CB1/CB2 receptor constructs we have been able to investigate the some of the structural features of the cannabinoid CB1 receptor implicated in initiating this pathway.
Section snippets
Drugs and chemicals
Forskolin and pertussis toxin were purchased from Sigma (Saint-Quentin-Fallavier, France). CP 55,940 {(−)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl) cyclohexan-1-ol} and SR 141716A {N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride} were synthesized by the Chemistry Department of Sanofi Recherche (Montpellier). Stock solutions of drugs were dissolved in dimethylsulfoxide at 10−2 M and stored at −20°C. The
Cannabinoid CB1 receptor-mediated cAMP accumulation
We first carried out a time course study of the effect of a single concentration of CP 55,940 on a CHO cell line permanently expressing the cannabinoid CB1 receptor or with CHO cells transiently expressing the receptor. The results presented in Fig. 1a and b show that the results were the same in both expression systems. The agonist at 10−6 M induced cAMP accumulation that quickly rose to a peak within 30 min, before falling slightly to a plateau where it remained for at least 4 h. As shown in
Discussion
Although there were early reports prior to cloning of the cannabinoid receptors that cannabinoids could stimulate cAMP accumulation (reviewed by Pertwee, 1988), it is only recently that some indications have appeared to suggest that the cannabinoid CB1 receptor can undergo dual G protein coupling (Maneuf and Brotchie, 1997; Bonhaus et al., 1998). In particular, it was reported (Maneuf and Brotchie, 1997) that the cannabimimetic WIN 55,212-2, in the absence of a Gs stimulant, could enhance basal
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