The endogenous
cannabinoid,
anandamide (
arachidonoylethanolamide), and the sleep-inducing factor,
oleamide (cis-9-octadecenoamide), represent two classes of long-chain
fatty acid amides with several neuronal actions and metabolic pathways in common. Here we report that these two compounds are present in human
breast carcinoma EFM-19 cells and rat adrenal
pheochromocytoma PC-12 cells, together with the
enzyme responsible for their degradation,
fatty acid amide hydrolase, and the proposed biosynthetic precursors for
arachidonoylethanolamide and related acylethanolamides, the N-acyl-
phosphatidylethanolamines.
Lipids extracted from cells labelled with [14C]
ethanolamine contained radioactive compounds with the same chromatographic behaviour as
arachidonoylethanolamide and acyl-PtdEtns. The levels of these compounds were not influenced by either stimulation with
ionomycin in EFM-19 cells or two-week treatment with the
nerve growth factor in PC-12 cells. The chemical nature of
arachidonoylethanolamide, related acylethanolamides and the corresponding acyl-PtdEtns was confirmed by gas chromatographic/mass spectrometric analyses of the purified compounds, which also showed the presence of higher levels of
oleamide. The latter compound, which does not activate the central
CB1 cannabinoid receptor, exhibited an anti-proliferative action on EFM-19 cells at higher concentrations than
arachidonoylethanolamide (IC50 = 11.3 microM for
oleamide and 2.1 microM for
arachidonoylethanolamide), while at a low, inactive dose it potentiated an
arachidonoylethanolamide cytostatic effect. The
CB1 receptor selective antagonist
SR 141716A (0.5 microM) reversed the effect of both
arachidonoylethanolamide and
oleamide. EFM-19 cells and PC-12 cells were found to contain a membrane-bound [14C]
arachidonoylethanolamide-hydrolysing activity with pH dependency and sensitivity to inhibitors similar to those previously reported for
fatty acid amide hydrolase. This
enzyme was inhibited by
oleamide in both intact cells and cell-free preparations. The presence of transcripts of
fatty acid amide hydrolase in these cells was shown by northern blot analyses of their total
RNA. The rate of [14C]
arachidonoylethanolamide hydrolysis by intact cells, the kinetic parameters of
arachidonoylethanolamide enzymatic hydrolysis and the amounts of the
fatty acid amide hydrolase transcript, were not significantly influenced by a two-week treatment with
nerve growth factor and subsequent transformation of PC-12 cells into neuron-like cells. These data show for the first time that: (a) induction by
nerve growth factor of a sympathetic neuronal phenotype in PC-12 cells has no effect on
arachidonoylethanolamide/
oleamide metabolism, (b)
arachidonoylethanolamide and
oleamide are
autacoid suppressors of human
breast cancer cell proliferation. Moreover these data lend conclusive support to the previous hypothesis that
oleamide may act as an enhancer of
arachidonoylethanolamide actions through competitive inhibition of its degradation.