Two types of cannabinoid receptor have been discovered so far, CB(1) (2.1: CBD:1:CB1:), cloned in 1990, and CB(2) (2.1:CBD:2:CB2:), cloned in 1993. Distinction between these receptors is based on differences in their predicted amino acid sequence, signaling mechanisms, tissue distribution, and sensitivity to certain potent agonists and antagonists that show marked selectivity for one or the other receptor type. Cannabinoid receptors CB(1) and CB(2) exhibit 48% amino acid sequence identity. Both receptor types are coupled through G proteins to adenylyl cyclase and mitogen-activated protein kinase. CB(1) receptors are also coupled through G proteins to several types of calcium and potassium channels. These receptors exist primarily on central and peripheral neurons, one of their functions being to inhibit neurotransmitter release. Indeed, endogenous CB(1) agonists probably serve as retrograde synaptic messengers. CB(2) receptors are present mainly on immune cells. Such cells also express CB(1) receptors, albeit to a lesser extent, with both receptor types exerting a broad spectrum of immune effects that includes modulation of cytokine release. Of several endogenous agonists for cannabinoid receptors identified thus far, the most notable are arachidonoylethanolamide, 2-arachidonoylglycerol, and 2-arachidonylglyceryl ether. It is unclear whether these eicosanoid molecules are the only, or primary, endogenous agonists. Hence, we consider it premature to rename cannabinoid receptors after an endogenous agonist as is recommended by the International Union of Pharmacology Committee on Receptor Nomenclature and Drug Classification. Although pharmacological evidence for the existence of additional types of cannabinoid receptor is emerging, other kinds of supporting evidence are still lacking.

Abstract

In the present study, we showed that Chinese hamster ovary (CHO) cells transfected with human central cannabinoid receptor (CB1) exhibit high constitutive activity at both levels of mitogen-activated protein kinase (MAPK) and adenylyl cyclase. These activities could be blocked by the CB1-selective ligand, SR 141716A, that functions as an inverse agonist. Moreover, binding studies showed that guanine nucleotides decreased the binding of the agonist CP-55,940, an effect usually observed with agonists, whereas it enhanced the binding of SR 141716A, a property of inverse agonists. Unexpectedly, we found that CB1-mediated effects of SR 141716A included inhibition of MAPK activation by pertussis toxinsensitive receptor-tyrosine kinase such as insulin or insulin-like growth factor 1 receptors but not by pertussis toxin-insensitive receptor-tyrosine kinase such as the fibroblast growth factor receptor. We also observed similar results when cells were stimulated with Mas-7, a mastoparan analog, that directly activates the G i protein. Furthermore, SR 141716A inhibited guanosine 5-0-(thiotriphosphate) uptake induced by CP-55,940 or Mas-7 in CHO-CB1 cell membranes. This indicates that, in addition to the inhibition of autoactivated CB1, SR 141716A can deliver a biological signal that blocks the G i protein and consequently abrogates most of the G i -mediated responses. By contrast, SR 141716A had no effect on MAPK activation by insulin or IGF1 in CHO cells lacking CB1 receptors, ruling out the possibility of a direct interaction of SR 141716A with the G i protein. This supports the notion that the G i protein may act as a negative intracellular signaling cross-talk molecule. From these original results, which considerably enlarge the biological properties of the inverse agonist, we propose a novel model for receptor/ligand interactions.

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Based on binding, functional, and pharmacological data, this study introduces SR147778 [5-(4-bromophenyl)-1-(2,4-dichlorophenyl)-4-ethyl-N-(1-piperidinyl)-1H-pyrazole-3-carboxamide] as a highly potent, selective, and orally active antagonist for the CB1 receptor. This compound displays nanomolar affinity (K i ϭ 0.56 and 3.5 nM) for both the rat brain and human CB1 recombinant receptors, respectively. It has low affinity (K i ϭ 400 nM) for both the rat spleen and human CB2 receptors. Furthermore, it shows no affinity for any of the over 100 targets investigated (IC 50 Ͼ 1 M). In vitro, SR147778 antagonizes the inhibitory effects of CP 55,940 [(1R,3R,4R)-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-4-(3-hydroxypropyl)cyclohexan-1-ol] on both the mouse vas deferens contractions (pA 2 value ϭ 8.1) and on forskolin-stimulated adenylyl cyclase activity in the U373 MG cell lines (pA 2 value ϭ 8.2) but not in Chinese hamster ovary (CHO) cells permanently expressing the human peripheral cannabinoid receptor (hCB2). SR147778 is able to block the mitogen-activated protein kinase activity induced by CP 55,940 in the CHO cell line expressing human brain cannabinoid receptor (IC 50 ϭ 9.6 nM) but was inactive in cells expressing hCB2. After oral administration, SR147778 displaced the ex vivo [3 H]-CP 55,940 binding to mouse brain membranes (ED 50 ϭ 3.8 mg/ kg) with a long duration of action, whereas it did not interact with the CB2 receptor expressed in the mouse spleen. Using different routes of administration, SR147778 (0.3-3 mg/kg) is shown to antagonize pharmacological effects (hypothermia, analgesia, and gastrointestinal transit) induced by R-(ϩ)-(2,3-dihydro-5-methyl-3-[{4-morpholinyl}methyl] pyrol [1,2,3-de]-1,4-benzoxazin-6-yl)(1-naphthalenyl) methanone in mice. Finally, per se, SR147778 (0.3-10 mg/kg) is able to reduce ethanol or sucrose consumption in mice and rats and food intake in fasted and nondeprived rats.Marijuana (Cannabis sativa L.) has been used for many centuries as a recreational drug or as a therapeutic agent. It is now well established that tetrahydrocannabinol (⌬ 9 -THC), the main active component of marijuana, the endocannabinoids (Devane et al., 1992;Mechoulam et al., 1995) as well as synthetic cannabinoid receptor agonists mediate their cellular effects through specific cannabinoid receptors, members of the G-protein-coupled receptor super family. To date, two cannabinoid receptors have been identified, CB1 (Matsuda et al., 1990; Gé rard et al., 1991) and CB2 (Munro et al., 1993). CB1 is predominantly expressed in the brain (Westlake et al., 1994) but also in some peripheral tissues (Gé rard et al., 1991;Bouaboula et al., 1993;Shire et al., 1995). The CB2 subtype is found mainly in immune cells (Munro et al., 1993; Galiè gue et al., 1995) but also in rat microglial cells (Walter et al., 2003). Both Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.104.067884.ABBREVIATIONS: ⌬ 9 -THC, tetrahydrocannabinol; SR147778, 5-(4-bromop...

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