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Cannabinoid receptors have been described in sea urchin sperm and shown to mediate inhibition of sperm acrosome reaction. Anandamide (arachidonoyl-ethanolamide), the mammalian physiological ligand at the cannabinoid CB1 receptor, has been subsequently found to effect this inhibition. Here we present data showing that ovaries from the sea urchin Paracentrotus lividus contain anandamide and two related acyl-ethanolamides, as well as enzymatic activities potentially responsible for their biosynthesis and degradation. Pilot experiments carried out with either ovaries or spermatozoa, extracted from both P. lividus and Arbacea lixula and radiolabelled with [14C]ethanolamine, showed that in sexually mature ovaries of both species significant levels of radioactivity were incorporated into a lipid component with the same chromatographic behaviour as anandamide. Lipid extracts from P. lividus ovaries were purified and analysed by gas chromatography/mass spectrometry which showed the presence of low but measurable amounts of anandamide, palmitoyl- and stearoyl-ethanolamides. The extracts were also found to contain lipid components with the same chromatographic behaviour as the N-acyl-phosphatidyl-ethanolamines, the phospholipid precursors of acyl-ethanolamides in mammalian tissues, and capable of releasing anandamide, palmitoyl- and stearoyl-ethanolamides upon digestion with S. chromofuscus phospholipase D. Accordingly, whole homogenates from P. lividus contained an enzymatic activity capable of converting synthetic [3H]N-arachidonoyl-phosphatidyl-ethanolamine into [3H]anandamide. Finally, mature ovaries of P. lividus were shown also to contain an amidohydrolase activity which catalyses the hydrolysis of anandamide and palmitoyl-ethanolamide to ethanolamine. This enzyme displayed subcellular distribution, pH/temperature dependency profiles and sensitivity to inhibitors similar but not identical to those of the previously described 'anandamide amidohydrolase' from mammalian tissues. These data support the hypothesis, formulated in previous studies, that anandamide or related metabolites may be oocyte-derived cannabimimetic regulators of sea urchin fertility.

The monoacylglycerol 2-arachidonoylglycerol (2-AG) has been recently suggested as a possible endogenous agonist at cannabinoid receptors both in brain and peripheral tissues. Here we report that a widely used model for neuronal cells, mouse N18TG2 neuroblastoma cells, which contain the CB1 cannabinoid receptor, also biosynthesize, release and degrade 2-AG. Stimulation with ionomycin (1-5 microM) of intact cells prelabelled with [3H]arachidonic acid ([3H]AA) led to the formation of high levels of a radioactive component with the same chromatographic behaviour as synthetic standards of 2-AG in TLC and HPLC analyses. The amounts of this metabolite were negligible in unstimulated cells, and greatly decreased in cells stimulated in the presence of the Ca2+-chelating agent EGTA. The purified component was further characterized as 2-AG by: (1) digestion with Rhizopus arrhizus lipase, which yielded radiolabelled AA; (2) gas chromatographic-MS analyses; and (3) TLC analyses on borate-impregnated plates. Approx. 20% of the 2-AG produced by stimulated cells was found to be released into the incubation medium when this contained 0.1% BSA. Subcellular fractions of N18TG2 cells were shown to contain enzymic activity or activities catalysing the hydrolysis of synthetic [3H]2-AG to [3H]AA. Cell homogenates were also found to convert synthetic [3H]sn-1-acyl-2-arachidonoylglycerols (AcAGs) into [3H]2-AG, suggesting that 2-AG might be derived from AcAG hydrolysis. When compared with ionomycin stimulation, treatment of cells with exogenous phospholipase C, but not with phospholipase D or A2, led to a much higher formation of 2-AG and AcAGs. However, treatment of cells with phospholipase A2 10 min before ionomycin stimulation caused a 2.5-3-fold potentiation of 2-AG and AcAG levels with respect to ionomycin alone, whereas preincubation with the phospholipase C inhibitor neomycin sulphate did not inhibit the effect of ionomycin on 2-AG and AcAG levels. These results suggest that the Ca2+-induced formation of 2-AG proceeds through the intermediacy of AcAGs but not necessarily through phospholipase C activation. By showing for the first time the existence of molecular mechanisms for the inactivation and the Ca2+-dependent biosynthesis and release of 2-AG in neuronal cells, the present paper supports the hypothesis that this cannabimimetic monoacylglycerol might be a physiological neuromodulator.

The endogenous cannabimimetic substance, anandamide (N-arachidonoyl-ethanolamine) and the recently isolated sleep-inducing factor, oleoyl-amide (cis-9,10-octadecenoamide), belong to two neuroactive fatty acid amide classes whose action in mammals has been shown to be controlled by enzymatic amide bond hydrolysis. Here we report the partial characterisation and purification of 'anandamide amidohydrolase' from membrane fractions of N18 neuroblastoma cells, and provide evidence for a further and previously unsuspected role of this enzyme. An enzymatic activity catalysing the hydrolysis of [14C]anandamide was found in both microsomal and 10,000 x g pellet fractions. The latter fractions, which displayed the highest Vmax for anandamide, were used for further characterisation of the enzyme, and were found to catalyse the hydrolysis also of [14C]oleoyl-amide, with an apparent Km of 9.0 +/- 2.2 microM. [14C]anandamide- and [14C]oleoyl-amide-hydrolysing activities: (i) exhibited identical pH- and temperature-dependency profiles; (ii) were inhibited by alkylating agents; (iii) were competitively inhibited by the phospholipase A2 inhibitor arachidonyl-trifluoromethyl-ketone with the same IC50 (3 microM); (iv) were competitively inhibited by both anandamide (or other polyunsaturated fatty acid-ethanolamides) and oleoyl-amide. Proteins solubilised from 10,000 x g pellets were directly analysed by isoelectric focusing, yielding purified fractions capable of catalysing the hydrolysis of both [14C]anandamide and [14C]oleoyl-amide. These data suggest that 'anandamide amidohydrolase' enzymes, such as that characterised in this study, may be used by neuronal cells also to hydrolyse the novel sleep-inducing factor oleoyl-amide.