Cis-9,10-octadecenoamide (
oleamide) was isolated from the cerebrospinal fluid of sleep-deprived mammals and shown to induce sleep in rats. The
enzyme catalyzing the hydrolysis of the
amide bond of
oleamide as well as of
anandamide, the putative endogenous
ligand of
cannabinoid receptors, was purified from rat liver, cloned, shown to be expressed also in brain and named
fatty acid amide hydrolase (FAAH). The enzymatic synthesis of
oleamide from
oleic acid and
ammonia by rat brain microsomes has been also described. However, no evidence has been reported so far on the neuronal origin of
oleamide, necessary in order to postulate for this compound a role as a
neuromodulator. Here we show for the first time that
oleamide is produced by a neuronal cell type and that its biosynthesis in intact neurons is not likely to occur through the direct condensation of
oleic acid and
ammonia. A
lipid metabolite was extracted and purified from mouse
neuroblastoma N18TG2 cells through a sequence of chromatographic steps and characterized as
oleamide by means of gas chromatography/electron impact mass spectrometry (GC/EIMS). The amount of
oleamide, as estimated by GC analyses carried out in comparison with known amounts of synthetic
oleamide, was 55.0+/-09.5 pmols/10(7) cells, compared to less than 0.7 pmol/10(7) cells for
anandamide in the same cells. When N18TG2 cells were prelabeled with [14C]
oleic acid and the
lipids extracted and purified, a radioactive component with the same chromatographic behavior as
oleamide was found whose levels: (1) were not significantly influenced by stimulation with
ionomycin; (2) were slightly increased by incubation with FAAH inhibitor phenyl-methyl-sulphonyl-
fluoride (PMSF); (3) appeared to correlate with [14C]
oleic acid incorporation into
phospholipids but not with free [14C]
oleic acid levels. N18TG2 cell membranes were shown to contain an enzymatic activity catalyzing the synthesis of
oleamide from
oleic acid and
ammonia. This activity was inhibited by FAAH selective inhibitors arachidonoyltrifluoromethylketone and
methylarachidonoylfluorophosphonate, as well as by an excess of
anandamide, and by PMSF at the same concentration which increased
oleamide formation in intact cells. These data suggest that a FAAH-like
enzyme working "in reverse" may be responsible for the formation of
oleamide in cell-free preparations but not in whole cells.