Abstract

Endocannabinoid (EC) levels are governed by the balance between synthesis, metabolism and trans-membrane transport. We studied the fasctors regulating intracellular and extracellular EC concentrations pharmacologically by examining different excitatory stimuli, URB597, an inhibitor of the major AEA catabolic enzyme: fatty acid amide hydrolase (FAAH) and VDM11; a potent and selective inhibitor of the anandamide membrane transporter; on intra- and extracellular ECs in a brain slice preparation.
Brain slices from male Lister hooded rats (>250 g) were prepared as previously described (Sarmad et al., 2007) and EC levels measured by LC/tandem mass spectrometry. (Richardson et al., 2007).
Depolarizing levels of KCl (50 mM) stimulated anandamide (AEA) and oleoylethanolamide (OEA ) synthesis in the tissue significantly whilst no changes were observed for 2-arachidonoyl glycerol (2-AG ) or palmitoyl-ethanolamide (PEA). Exposure to excitatory amino acid (glutamate) or carbachol had no significant effect on EC synthesis. Blockade of FAAH by URB597 (1μM) however significantly increased AEA, OEA and PEA levels but had no effect on 2-AG. VDM11 did not affect EC tissue levels.
The effects of the other drugs on extracellular ECs mirrored tissue changes with the exception that VDM11 managed to increase the release of AEA, OEA and PEA non significantly, with no effect on release of 2-AG but when combined with URB597 increased 2-AG release. We previously showed that URB597 was not able to produce this effect when administered alone.
The data provide no evidence for tonic regulation of synthesis by CB1 or TRPV1 receptors. The effects of URB597 suggest a high turnover rate of AEA, PEA and OEA in this preparation and that 2-AG is not subject to catabolism by FAAH. The release of ECs from cortical slices appears, in the main, to be passively driven by the trans-membrane concentration gradient.
Refrences:
Richardson D et al. (2007)Anal Biochem.360;216-226.
Sarmad S et al.. (2007) Proceedings of the BPS, Oxford Meeting, P86