Effects of sustained inhibition of FAAH on endocannabinoid synthesis and catabolism in the midbrain and liver

Bright Okine, Leonie Norris, Annie Patel, Peter Christensen, Stephen Alexander, David Kendall, David Barrett, Andrew Bennett, Victoria Chapman

University of Nottingham, Nottingham, UK

Fatty acid amide hydrolase (FAAH) is an intracellular serine hydrolase enzyme which hydrolyses fatty acid ethanolamides (FAEs) including anandamide (AEA) and N-palmitoylethanolamine (PEA), produced primarily by the action of N-acylphosphatidylethanolamine phospholipase D (NAPE-PLD). Acute inhibition of FAAH by the irreversible inhibitor URB597 increases levels of AEA and produces an analgesic phenotype (Jhaveri et. al., 2008). The impact of sustained inhibition of FAAH on endocannabinoid turnover is unknown. Herein, the effects of URB597 on levels of FAAH, NAPE PLD (FAE synthetic enzyme), and MAGL (monoacylglycerol lipase, which hydrolyses the endocannabinoid 2-arachidonoylglycerol, 2-AG) mRNA and protein expression, FAAH enzyme activity and levels of AEA, PEA and 2-AG are reported.

Adult male Sprague-Dawley rats (200-250g) received intraperitonial injections of URB597 (0.3 mg kg-1) or vehicle on four consecutive days. Rats were killed by decapitation on day 4 and midbrain and liver samples were collected and snap frozen in liquid nitrogen. FAAH, NAPE-PLD and MAGL mRNA in each tissue was determined, relative to β-actin, using Taqman-based real-time PCR, while protein levels were assayed using immunoblotting. FAAH activity (measured as the hydrolysis of 100 μM oleamide) and levels of AEA, PEA and 2AG were measured using a modified analytical method based on previous work (Jhaveri et al., 2008). Statistical analysis on data from 5-6 rats was conducted using 1-way ANOVA with Bonferonni’s multiple comparison test (for FAAH activity data) unpaired T-test (for mRNA and protein data) or Mann-Whitney U test (for endocannabinoid data).

4 day dosing with URB597 significantly elevated levels of PEA in the midbrain and liver (P<0.01 for both). Levels of AEA in the liver and midbrain were unquantifiable. Levels of 2-AG were unaltered following URB597 treatment in either the midbrain or liver. URB597 treatment significantly decreased FAAH activity in the midbrain (0.16 ± 0.06 nmol min-1 mg-1 protein, P<0.05) and liver (0.29 ± 0.04, P<0.001) compared to vehicle-treated rats (0.97 ± 0.32 and 1.20 ± 0.13, respectively). URB597 treatment significantly reduced FAAH mRNA expression in the midbrain (0.46 ± 0.02,P<0.05), compared with vehicle treated rats (0.75 ± 0.11). By contrast, FAAH protein levels in the midbrain were unaltered by URB597 treatment. FAAH mRNA and protein were unaltered in the liver of URB597-treated rats, compared to controls. NAPE-PLD and MAGL mRNA and protein levels were unaltered in the midbrain or liver of URB597-treated rats, compared to vehicle-treated rats.

Following repeated URB597 treatment, the marked inhibition of FAAH activity in both the liver and midbrain is consistent with the elevation of FAEs in these tissues. The decrease in FAAH mRNA in the midbrain suggests that sustained inhibition of FAAH is associated with adaptive changes in the gene expression of FAAH, but not the other enzymes involved in endocannabinoid turnover.

This work is supported by the MRC ( B.O, L.N, A.P) and BBSRC (P.C)

Jhaveri et. al., (2008) Neuropharmacology 55, 85-93