Alternative pathways of anandamide biosynthesis in rat testes

We have investigated the biosynthesis long-chain N-acylethanolamines (NAEs) from endogenous substrates in rat testes membranes with special emphasis on anandamide (20:4n-6 NAE), a cannabinoid receptor agonist. Incubation of various membrane preparations with 5 mM Ca2+ produced both N-acyl phosphatidylethanolamine (N-acyl PE) and NAE with primarily (similar to 85%) N-palmitoyl groups (16:0 NAE) and less than 2% 20:4n-6 NAE. In contrast, incubation of these membranes with 5 mM EGTA and 10 mM ethanolamine had little effect on N-acyl PE composition but yielded NAEs whose major constituent (32-37%) was anandamide. Incubations with [1,1,2,2,-H-2(4)]ethanolamine in media containing 40% (H2O)-O-18 showed that the Ca2+-independent NAE synthesis occurred by direct condensation of ethanolamine with free fatty acids present in the membrane preparation. This biosynthetic activity occurred at ethanolamine concentrations as low as 50 mu M and exhibited substrate selectivity for arachidonate which increased with increasing ethanolamine concentrations. The results of inhibitor experiments suggest that the Ca2+-independent NAE synthesis was catalyzed by the NAE amidohydrolase acting in reverse. This condensation reaction could be important in agonist-induced anandamide synthesis for cell signalling through cannabinoid receptors. (C) 1998 Elsevier Science Ireland Ltd. All rights reserved.