Evaluation of the role of the arachidonic acid cascade in anandamide's in vivo effects in mice
Introduction
Arachidonylethanolamide (anandamide), an endogenous ligand for the cannabinoid receptor, binds competitively to brain cannabinoid (CB1) receptors and shares many, but not all, of the in vivo effects of Δ9-tetrahydrocannabinol (Δ9-THC; Smith et al., 1994). The in vivo effects of exogenously administered anandamide in mice include characteristic cannabinoid effects of hypomobility, antinociception, hypothermia, and catalepsy (Smith et al., 1994). While other classes of compounds (e.g., CNS depressants, antipsychotics, and transient receptor potential vanilloid type 1 (TRPV1) agonists) may produce one or more of these effects (Di Marzo et al., 2002, Wiley and Martin, 2003), traditional tetrahydrocannabinol cannabinoids and synthetic bicyclic and aminoakylindole cannabinoids consistently and dose-dependently produced all four and their potency in doing so was strongly and positively correlated with their CB1 binding affinity (Compton et al., 1993, Martin et al., 1991). Further, the cannabinoid effects of Δ9-THC and these classes of synthetic cannabinoids were blocked by N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride (SR141716A), an antagonist selective for CB1 receptors (Rinaldi-Carmona et al., 1994). In contrast, the effects of non-cannabinoid compounds in the tetrad were not affected by SR141716A (Wiley and Martin, 2003). Anandamide-induced effects also were not reversed by SR141716A (Adams et al., 1998). At least two possible explanations may account for the failure of SR141716A to block the in vivo cannabinoid effects of anandamide: (1) direct anandamide activity at non-CB1 receptors and/or (2) rapid degradation of anandamide to metabolites that are active in vivo through non-CB1 mechanisms. These pharmacodynamic and pharmacokinetic explanations are not mutually exclusive; however, in this study, we have focused exclusively on evaluation of the role of anandamide metabolism on its in vivo pharmacology.
One major difference between anandamide and classical exogenous cannabinoids is that, unlike long-acting plant-derived cannabinoids, anandamide has a comparatively short duration of action. Termination of anandamide action may be effected through its rapid metabolism to inactive metabolites (Deutsch and Chin, 1993) and through uptake by a putative anandamide transporter (Beltramo et al., 1997). The rapid metabolic inactivation of anandamide through hydrolysis is mediated by action of fatty acid amide hydrolase (Cravatt et al., 1996, Deutsch and Chin, 1993), with arachidonic acid as the primary metabolite (Wiley et al., 2000, Willoughby et al., 1997). Anandamide may also undergo oxidative metabolism through interaction of its arachidonyl substituent with enzymes in the arachidonic acid cascade (Fig. 1) (see Kozak and Marnett, 2002 for review). Under physiological conditions, arachidonic acid is bound to phospholipids within the cell membrane and is released, as needed, through the cleaving action of a phospholipase enzyme. Free arachidonic acid is metabolized through three primary metabolic pathways: (1) the action of cyclooxygenase enzymes 1 and 2 (constitutive and inducible, respectively), resulting in formation of thromboxane A2 and prostaglandins, (2) metabolism by lipoxygenase enzymes, recsulting in formation of leukotrienes and (3) metabolism by cytochrome P450 to epoxyeicosatrienoic acids or dihydroxyacids. Given the similarity of the chemical structures of anandamide and arachidonic acid (Fig. 2), we hypothesize that oxidation of anandamide may be mediated by some of the same enzymes. For example, anandamide is selectively oxidized by cyclooxygenase-2 to prostaglandin E2 ethanolamide (Yu et al., 1997) and other prostacyclin ethanolamides (Kozak et al., 2002). Anandamide is also sensitive to oxidation by 12- and 15-lipoxygenase (Ueda et al., 1995a, Ueda et al., 1995b) and perhaps by cytochrome P450 (Capdevila and Falck, 2001), although this latter pathway has received limited research attention. The physiological relevance of the resulting end products is unclear and is under active investigation by a number of research groups (see Kozak and Marnett, 2002 for review).
In order to examine the role of oxidative metabolism of anandamide to arachidonic acid in producing its in vivo effects, we disrupted this process through administration of pharmacological agents that blocked one or more steps of the arachidonic acid pathway. First, we evaluated the in vivo effects of anandamide by inhibiting its metabolism to arachidonic acid by administering phenylmethyl sulfonyl fluoride (PMSF), a non-specific and irreversible amidase inhibitor (Childers et al., 1994, Deutsch and Chin, 1993). Subsequently, we evaluated the effects of selected pharmacological modulators of the arachidonic acid pathway on anandamide-induced effects in the tetrad tests. These modulators included non-steroidal anti-inflammatories and non-selective cyclooxygenase inhibitors (ibuprofen, aspirin, and indomethacin), acetaminophen (alters cyclooxygenase metabolism of arachidonic acid in an undetermined manner; Lucas et al., 2005), dexamethasone (glucocorticoid and phospholipase inhibitor), esculetin (5- and 12-lipoxygenase inhibitor, Neichi et al., 1983), nimesulide (selective cyclooxygenase-2 inhibitor, Pairet and van Ryn, 1998), and phenidone (dual lipoxygenase–cyclooxygenase inhibitor, Flynn et al., 1986).
Section snippets
Subjects
Naïve male ICR mice (25–32 g), purchased from Harlan (Dublin, VA), were housed in groups of five. All animals were kept in a temperature-controlled (20–22 °C) environment with a 12-h light–dark cycle (lights on at 7 a.m.). Separate mice were used for testing each drug dose in the in vivo behavioral procedures. The mice received food and water ad libitum.
Apparatus
Measurement of spontaneous activity in mice occurred in plastic mouse cages (without bedding) surrounded by panels of photocell beams (Digiscan
Results
Anandamide (10 mg/kg) produced characteristic cannabinoid effects in all of the tetrad tests and these effects were not reversed by 3 mg/kg SR141716A (Fig. 3). These findings represent a replication of our previous results showing that anandamide's effects in the tetrad were not blocked by SR141716A (Adams et al., 1998). Co-administration of 30 mg/kg PMSF and 10 mg/kg anandamide also produced all four cannabinoid effects (i.e., significant decreases in spontaneous activity and rectal
Discussion
The goal of this study was to determine the degree to which metabolism of anandamide via the arachidonic acid pathway might contribute to anandamide's in vivo pharmacological effects in the tetrad model and their lack of reversal by SR141716A. While the tetrad tests are relatively selective for cannabinoids when used as a battery of tests (versus individually) (Martin et al., 1991), they are not pharmacologically specific and non-cannabinoid compounds may produce similar effects (e.g., CNS
Acknowledgments
Research supported by National Institute on Drug Abuse research grants DA-09789 and DA-08904.
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