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.

Fig. 1.

Anandamide transport in primary cultures of FAAH^–/– neurons. (a)[³H]Anandamide hydrolysis in homogenates of brain neurons from wild-type mice (open bars) and FAAH^–/– mice (filled bars) (mean ± SEM, n = 6). (b) Accumulation of [³H]anandamide in wild-type and FAAH^–/– neurons is temperature-dependent (mean ± SEM, n = 4). (c) Accumulation of [³H]anandamide in wild-type (open bars) and FAAH^–/– (filled bars) neurons is inhibited by nonradioactive anandamide (AEA, 100 μM) or AM404 (10 μM) (*, P < 0.05, **, P < 0.01, ***, P < 0.001; n = 3). Time course of [³H]anandamide accumulation in wild-type (+/+) (d) and FAAH^–/– (–/–)(e) neurons in the absence or presence of AM404 (10 μM) (hatched bars) (**, P < 0.01, ***, P < 0.001 vs. control; ##, P < 0.01 vs. 0 time point; n = 3).

Fig. 2.

Administration of AM404 enhances the effects of exogenous anandamide in both wild-type and FAAH^–/– mice. AM404 increases anandamide-induced hypothermia in wild-type (+/+) mice (a)(▪, vehicle; •, anandamide, 5 mg/kg; ▴, AM404, 10 mg/kg; ▾, AM404 plus anandamide) and FAAH^–/– (–/–) mice (b) (▪ vehicle; □ anandamide, 2 mg/kg; ▴ AM404, 7 mg/kg; ▾ AM404 plus anandamide) (*, P < 0.05, **, P < 0.01, ***, P < 0.001 vs. vehicle; #, P < 0.05, ##, P < 0.01 vs. anandamide alone; n = 9). (c) The CB₁ antagonist rimonabant (Rim, 0.3 mg/kg, i.p.) but not the vanilloid antagonist capsazepine (Cap, 30 mg/kg, i.p), prevents the combined effect of AM404 (7 mg/kg) plus anandamide (AEA, 2 mg/kg) in FAAH^–/– mice (*, P < 0.05; mean ± SEM). (d) AM404 levels after a 30-min incubation with brain membranes of wild-type (open bars) or FAAH^–/– (filled bars) mice. **, P < 0.01; ***, P < 0.001; n = 5; mean ± SEM.

Fig. 3.

Endogenous levels of anandamide (AEA), oleoylethanolamide (OEA), and palmitoylethanolamide (PEA) in the brains of wild-type mice 1 h after administration of AM404 (10 mg/kg, i.p.). **, P < 0.01; n = 5.

Fig. 4.

AM1172, a metabolically stable inhibitor of anandamide transport. Chemical structures of AM1172 (a) and AM404 (b). Note the reverse-amide moiety in AM1172, which may confer stability toward FAAH hydrolysis. (c) AM1172 levels after a 30-min incubation with brain membranes from wild-type (open bars) or FAAH^–/– (filled bars) mice. (d) AM1172 (▿) does not inhibit FAAH activity in rat brain membranes; also shown are the effects of AM404 (▴) and the FAAH inhibitors URB597 (□) and AM374 (▪). Results are from one experiment, performed in triplicate, representative of three.

Fig. 5.

Effects of AM1172 and AM404 (0–10 μM) on [³H]anandamide transport in human astrocytoma cells (a) and rat cortical neurons (b) in culture. Time course of [³H]anandamide accumulation in neurons from wild-type mice (+/+)(c) or FAAH^–/– (d)(–/–) mice in the absence or presence of AM1172 (10 μM) (hatched bars). **, P < 0.01; ***, P < 0.001 vs. control; n = 4.