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Assessment of anandamide's pharmacological effects in mice deficient of both fatty acid amide hydrolase and cannabinoid CB1 receptors

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Abstract

In the present study, we investigated whether anandamide produces its behavioral effects through a cannabinoid CB1 receptor mechanism of action. The behavioral effects of anandamide were evaluated in mice that lacked both fatty acid amide hydrolase (FAAH) and cannabinoid CB1 receptors (DKO) as compared to FAAH (−/−), cannabinoid CB1 (−/−), and wild type mice. Anandamide produced analgesia, catalepsy, and hypothermia in FAAH (−/−) mice, but failed to elicit any of these effects in the other three genotypes. In contrast, anandamide decreased locomotor behavior regardless of genotype, suggesting the involvement of multiple mechanisms of action, including its products of degradation. These findings indicate that the cannabinoid CB1 receptor is the predominant target mediating anandamide's behavioral effects.

Introduction

While anandamide is most prominently known as the first endogenous cannabinoid extracted from mammalian brain, it has activity at multiple targets including cannabinoid CB1 receptors, cannabinoid CB2 receptors (Calignano et al., 1998, Sokal et al., 2003), TRPV1 receptors (Tognetto et al., 2001, Zygmunt et al., 1999), potassium channels (Maingret et al., 2001), T-type calcium channels (Chemin et al., 2001), and gap junctions (Venance et al., 1995). The rapid catabolism and consequent short half-life (Willoughby et al., 1997) of this lipid signaling molecule by fatty acid amide hydrolase (FAAH) have undoubtedly contributed to discrepant findings from various investigations in whole animals.(Adams et al., 1998, Calignano et al., 1998, Costa et al., 1999, Di Marzo et al., 2000, Farquhar-Smith and Rice, 2001, Jaggar et al., 1998, Lichtman et al., 1996, Richardson et al., 1998a, Richardson et al., 1998b, Smith et al., 1998, Smith et al., 1994). Thus, anandamide's weak and transient pharmacological effects in mice (Smith et al., 1994), rats (Lichtman et al., 1996), and dogs (Lichtman et al., 1998) after exogenous administration is not altogether surprising. CB1 (−/−) mice as well as mice pretreated with the cannabinoid CB1 receptor antagonist rimonabant exhibit pharmacological effects following intravenously injected anandamide, suggesting a non-CB1 receptor mechanism of action (Adams et al., 1998, Di Marzo et al., 2000, Smith et al., 1994). In contrast, anandamide given through the intraperitoneal route of administration to either FAAH (−/−) mice or mice treated with FAAH inhibitors elicits profound pharmacological effects, which are blocked by SR141716 (Cravatt et al., 2001, Cravatt et al., 2004, Kathuria et al., 2003, Lichtman et al., 2004). Of course, a major difference between the two strategies is that high doses of anandamide were required to elicit a pharmacological effect in wild type mice and considerable quantities of metabolites are formed, whereas low doses of anandamide can produce pharmacological effects in FAAH (−/−) mice. While products of metabolism could contribute to anandamide's effects in the wild type animals, it remains plausible that the effects of this endogenous cannabinoid are mediated through a non-CB1 receptor mechanism of action.

The purpose of the present study was to investigate whether intact anandamide produces overt behavioral effects at sites other than cannabinoid CB1 receptors. In order to address this question, we created mice that lacked both FAAH and cannabinoid CB1 receptor genes (DKO mice) by selectively breeding FAAH (−/−) mice with CB1 (+/−) mice. We then investigated the effects of anandamide in DKO mice as compared to wild type, CB1 (−/−), and FAAH (−/−) mice in the tetrad test, a well established screen for cannabinoid activity (Compton et al., 1990, Compton et al., 1993, Martin et al., 1991).

Section snippets

Subjects

The subjects consisted of male and female mice on a C57BL/6 background of the following four genotypes: 1) wild type: FAAH (+/−)/CB1 (+/+); 2) CB1 (−/−) mice: FAAH (+/−)/CB1 (−/−); 3) FAAH (−/−) mice: FAAH (−/−)/CB1 (+/−); and 4) DKO: FAAH (−/−)/CB1 (−/−). Previous studies have demonstrated that FAAH (+/−) mice display virtually identical phenotypes as wild type mice (Cravatt et al., 2001, Cravatt et al., 2004, Lichtman et al., 2004) and thus were used as a control in this study. All subjects

Results

No relevant differences were found among the different genotypes with respect to baseline hot plate latencies or body temperatures (Table 1), though mice lacking cannabinoid CB1 receptors displayed a small, but a significant decrease in baseline rectal temperature, F(1, 39) = 7.6, P < 0.01. Significant three-way interactions were found among FAAH genotype, CB1 genotype, and anandamide for analgesia (F(1,39) = 37, P < 0.001), catalepsy (F(1,39) = 10, P < 0.01), and hypothermia (F(1,39) = 26, P < 0.001). As

Discussion

As previously found (Cravatt et al., 2001), anandamide produced analgesia, catalepsy, and hypothermia in genetically modified mice that lack FAAH, but here we report that it had no significant effects in wild type (i.e., FAAH (+/−) and CB1 (+/+)), CB1 (−/−) mice, and DKO mice. Thus, these effects of anandamide are regulated by FAAH and mediated through a cannabinoid CB1 receptor mechanism of action. Corroborating these findings is previous work demonstrating that rimonabant blocks each of these

Acknowledgement

This research was supported by the following grants from the NIH: P50DA005274, R01DA015197, R01DA015683, P01DA009789, and T32DA007027.

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