Pharmacological characterisation of endocannabinoid degradation and uptake in rat brain slices

Sarir Sarmad1, David Barrett2, Charles Marsden1, David Kendall1. 1Schools of Biomedical Sciences, Nottingham, United Kingdom, 2School of Pharmacy, Nottingham, United Kingdom.

A variety of proteins are known to play roles in synthesis, metabolism and trans-membrane transport of endocannabinoids (ECs). Although the exact mechanisms involved in EC uptake and degradation are a matter of debate, several compounds have been reported to inhibit selectively anandamide (AEA) uptake into the cell or AEA degradation by fatty acid amide hydrolase (FAAH). Pharmacological manipulation provides a key to the elucidation of the roles played by ECs. We examined the effects of PMSF (phenylmethylsulfonyl fluoride, a non-specific serine protease inhibitor), URB597 (3´-carbamoyl-biphenyl-3-yl-cyclohexyl carbamate) and MAFP (methyl arachidonoyl fluorophosphonate) irreversible and selective FAAH inhibitors, as well as VDM11 (a potent and selective inhibitor of the AEA membrane transport, AMT inhibitor) on intra- and extracellular ECs in a brain slice preparation.

Slices were prepared from the cerebral cortices of male Lister hooded rats (>250 g, n=6 throughout) as described before (Sarmad et al., 2007). Following organic solvent and solid phase extraction, EC levels were measured by liquid-chromatography-tandem mass spectrometry (Richardson et al. 2007). All drugs were dissolved in ethanol to a stock concentration of 10-2M, with subsequent dilution in KHB. Statistical analysis (Student’s t-test) compared basal levels with those following drug exposure. Data are expressed as mean±S.E.M. Previously, we showed that blockade of FAAH by URB597 significantly increased AEA, oleoylethanolamide (OEA) and palmitoylethanolamide (PEA) levels in the slices but had no effect on 2-arachidonoyl glycerol (2-AG). The increased EC levels due to URB597 were not altered in the presence of the CB1 receptor antagonist (SR141617A, 1μM) or the TRPV1 antagonist, capsazepine (1μM) or VDM11 (1,5,10 μM). We also found that URB597 exerted its effect in a calcium-independent manner; removal of extracellular Ca 2+ tended to elevate basal and URB597-stimulated tissue levels of AEA.

PMSF (100 μM) significantly increased AEA, OEA and PEA levels in slices by 2.09±0.21 (P=0.0008), 1.7±0.11 (P=0.0018) and 1.28±0.05 (P=0.0012) fold respectively. PMSF significantly lowered 2-AG levels in slices to 0.37±0.05 fold of basal (P=0.041). MAFP (10 μM) significantly increased AEA and OEA levels in tissues by 2.68±0.54 fold (P=0.0274) and 2.3±0.42 fold (P=0.0162), respectively. PEA levels were not altered significantly (1.69±0.23 fold, P=0.051), and had no effect on 2-AG. VDM11did not affect EC tissue levels.

EC levels detected in the medium mirrored tissue changes with the exception that VDM11 increased the release of AEA, OEA and PEA by 1.5 fold, with no effect on release of 2-AG.

VDM11, when combined with URB597, potentiated 2-AG release.

The release of ECs from cortical slices appears, in the main, to be passively driven by the trans-membrane concentration gradient. The effects of URB597 (the most potent), MAFP and PMSF suggest a high turnover rate of AEA, PEA and OEA in this preparation, but whether they exert their effect merely by inhibition of FAAH and/or other mechanisms needs further investigation.

Richardson D. et al. (2007) Anal Biochem. 360; 216-226

Sarmad S. et al., 2007 Proc BPS Oxford.