Anandamide transport is independent of fatty-acid amide hydrolase activity and is blocked by the hydrolysis-resistant inhibitor AM1172

Abstract

The endogenous cannabinoid anandamide is removed from the synaptic space by a high-affinity transport system present in neurons and astrocytes, which is inhibited by N-(4-hydroxyphenyl)-arachidonamide (AM404). After internalization, anandamide is hydrolyzed by fatty-acid amide hydrolase (FAAH), an intracellular membrane-bound enzyme that also cleaves AM404. Based on kinetic evidence, it has recently been suggested that anandamide internalization may be mediated by passive diffusion driven by FAAH activity. To test this possibility, in the present study, we have investigated anandamide internalization in wild-type and FAAH-deficient (FAAH ؊/؊ ) mice. Cortical neurons from either mouse strain internalized [ 3 H]anandamide through a similar mechanism, i.e., via a rapid temperature-sensitive and saturable process, which was blocked by AM404. Moreover, systemic administration of AM404 to either wild-type or FAAH ؊/؊ mice enhanced the hypothermic effects of exogenous anandamide, a response that was prevented by the CB 1 cannabinoid antagonist rimonabant (SR141716A). The results indicate that anandamide internalization in mouse brain neurons is independent of FAAH activity. In further support of this conclusion, the compound N-(5Z, 8Z, 11Z, 14Z eicosatetraenyl)-4-hydroxybenzamide (AM1172) blocked [ 3 H]anandamide internalization in rodent cortical neurons and human astrocytoma cells without acting as a FAAH substrate or inhibitor. AM1172 may serve as a prototype for novel anandamide transport inhibitors with increased metabolic stability.

Anxiolytic-Like Properties of the Anandamide Transport Inhibitor AM404

“…Tissues were immediately frozen on dry ice and stored at À801C until analysis. We extracted endocannabinoids and related lipids with methanol-chloroform, fractionated them by open-bed silica gel chromatography and quantified them by isotope-dilution liquid chromatography/mass spectrometry (LC/MS), as described by Fegley et al (2004).…”

The endogenous cannabinoid system and the treatment of marijuana dependence

“…However, the result may also be explained on purely technical grounds, as the high concentration of serum albumin used in the experiments of Glaser and collaborators was previously shown to prevent [ 3 H]anandamide internalization (Di Marzo et al, 1994;Hillard and Jarrahian 2003). Consistent with this interpretation, recent studies have provided additional evidence for the existence of an anandamide transport system independent of FAAH (Ligresti et al, 2004;Fegley et al, 2004). In particular, one of these studies has shown that cultures of cortical neurons isolated from the brain of FAAHnull mice internalize anandamide as efficiently as do neurons that express normal levels of the enzyme.…”

“…This implies that AM404 is not in fact a FAAH inhibitor, as it has been proposed (Glaser et al, 2003), but a FAAH substrate. In support of this idea, it was found that membranes prepared from the brains of normal mice rapidly hydrolyze AM404, whereas those prepared from mice that lack FAAH are unable to carry out this reaction (Fegley et al, 2004). The fact that FAAH is not directly involved in anandamide internalization (Ligresti et al, 2004;Fegley et al, 2004) raises the question of what mechanism provides the driving force for this process.…”

“…In support of this idea, it was found that membranes prepared from the brains of normal mice rapidly hydrolyze AM404, whereas those prepared from mice that lack FAAH are unable to carry out this reaction (Fegley et al, 2004). The fact that FAAH is not directly involved in anandamide internalization (Ligresti et al, 2004;Fegley et al, 2004) raises the question of what mechanism provides the driving force for this process. One possibility is that an intracellular protein may sequester anandamide at the membrane, driving its internalization and facilitating its movement to the mitochondria and the endoplasmic reticulum, where FAAH is primarily localized (Gulyas et al, 2004).…”

“…AM404 increases endogenous anandamide levels in brain tissue and peripheral blood of rats and mice (Fegley et al, 2004;Giuffrida et al, 2000). This effect is accompanied by a series of behavioral responses that, though blocked by the CB 1 antagonist rimonabant (SR141716A), are clearly distinguishable from those of direct cannabinoid agonists.…”