No abstract

Saturated and unsaturated N-acylethanolamines (NAEs) occur in desiccated seeds primarily as 16C and 18C species with N-palmitoylethanolamine and N-linoleoylethanolamine (NAE 18:2) being most abundant. Here, we examined the metabolic fate of NAEs in vitro and in vivo in imbibed cotton (Gossypium hirsutum) seeds. When synthetic [1-14 C]Npalmitoylethanolamine was used as a substrate, free fatty acids (FFA) were produced by extracts of imbibed cottonseeds. When synthetic [1-14 C]NAE 18:2 was used as a substrate, FFA and an additional lipid product(s) were formed. On the basis of polarity, we presumed that the unidentified lipid was a product of the lipoxygenase (LOX) pathway and that inclusion of the characteristic LOX inhibitors nordihydroguaiaretic acid and eicosatetraynoic acid reduced its formation in vitro and in vivo. The conversion of NAE 18:2 in imbibed cottonseed extracts to 12-oxo-13-hydroxy-N-(9Z)-octadecanoylethanolamine was confirmed by gas chromatography-mass spectrometry, indicating the presence of 13-LOX and 13-allene oxide synthase, which metabolized NAE 18:2. Cell fractionation studies showed that the NAE amidohydrolase, responsible for FFA production, was associated mostly with microsomes, whereas LOX, responsible for NAE 18:2-oxylipin production, was distributed in cytosol-enriched fractions and microsomes. The highest activity toward NAE by amidohydrolase was observed 4 to 8 h after imbibition and by LOX 8 h after imbibition. Our results collectively indicate that two pathways exist for NAE metabolism during seed imbibition: one to hydrolyze NAEs in a manner similar to the inactivation of endocannabinoid mediators in animal systems and the other to form novel NAE-derived oxylipins. The rapid depletion of NAEs by these pathways continues to point to a role for NAE metabolites in seed germination.In mammalian cells, N-acylethanolamines (NAEs) have varied physiological roles. N-Arachidonylethanolamine (anandamide), a type of NAE in mammalian brain tissue, is an endogenous ligand for the cannabinoid receptor and modulates neurotransmission. Anandamide also can activate vanilloid (capsaicin) receptors and function as an endogenous analgesic (Pertwee, 2001), and appears to be involved in neuroprotection (Hansen et al., 2000;Van der Stelt et al., 2001). In other animal tissues, NAEs have been implicated in immunomodulation (Buckley et al., 2000), synchronization of embryo development (Paria and Dey, 2000), and induction of apoptosis (Sarker et al., 2000). These endogenous bioactive molecules termed "endocannabinoids" are hydrolyzed by fatty acid amidohydrolase (AHase) to terminate their signaling functions.In plants NAEs are present in substantial amounts in desiccated cotton (Gossypium hirsutum) seeds (1.6 g Ϫ1 g fresh weight), and their levels decline after a few hours of imbibition . Individual NAEs were identified predominantly as 16C and 18C species with N-palmitoylethanolamine (NAE 16:0) and N-linoleoylethanolamine (NAE 18:2) being the most abundant. NAEs in both plant and animal cells are deri...

N-Acylethanolamines (NAEs) constitute a new class of plant lipids and are thought to play a role in plant defense strategies against pathogens. In plant defense systems, oxylipins generated by the lipoxygenase pathway are important actors. To date, it is not known whether plants also use endogeneous oxylipins derived from NAEs in their defense reactions. We tested whether members of the NAE class can be converted by enzymes constituting this pathway, such as (soybean) lipoxygenase-1, (alfalfa) hydroperoxide lyase and (flax seed) allene oxide synthase. We found that both a-N-linolenoylethanolamine and g-N-linolenoylethanolamine (18:3), as well as a-N-linolenoylamine and g-N-linolenoylamine were converted into their (13S )-hydroperoxide derivatives by lipoxygenase. Interestingly, only the hydroperoxides of a-N-linolenoyl(ethanol)amines and their linoleic acid analogs (18:2) were suitable substrates for hydroperoxide lyase. Hexanal and (3Z )-hexenal were identified as volatile products of the 18:2 and 18:3 fatty acid (ethanol)amides, respectively. 12-Oxo-N-(9Z )-dodecenoyl(ethanol)amine was the nonvolatile hydrolysis product. Kinetic studies with lipoxygenase and hydroperoxide lyase revealed that the fatty acid ethanolamides were converted as readily or even better than the corresponding free fatty acids. Allene oxide synthase utilized all substrates, but was most active on (13S )-hydroperoxy-a-N-linolenoylethanolamine and the (13S )-hydroperoxide of linoleic acid and its ethanolamine derivative. a-Ketols and g-ketols were characterized as products. In addition, cyclized products, i.e. 12-oxo-N-phytodienoylamines, derived from (13S )-hydroperoxy-a-N-linolenoylamines were found. The results presented here show that, in principle, hydroperoxide NAEs can be formed in plants and subsequently converted into novel phytooxylipins.

Anandamide, a novel neurotransmitter, has been reported to be dioxygenated by brain lipoxygenase [1, 11]. Anandamides constitute a new class of neuroregulatory fatty acid amides. However, little is known about the enzymatic dioxygenation of these lipids. Therefore, we have tested several members of the neuroactive fatty acid amide class containing a 1Z,4Z‐pentadiene system whether they could be dioxygenated by soybean lipoxygenase‐1, which is a model enzyme for mammalian lipoxygenases. In this study it was found that lipoxygenase‐1 converts N‐linoleoylethanolamide (ODNHEtOH), N‐linoleoylamide (ODNH2), N‐linoleoylmethylamide (ODNHMe) and N,N‐linoleoyldimethylamide (ODN(Me)2 into 13‐(S)‐hydroperoxy‐9Z,11E‐octadeca‐9,11‐dienoyl amides derivatives. The apparent K m values for ODNHEtOH (23.6±3.7 μM), ODNH2 (8.60±0.65 μM) and linoleic acid (OD: 8.85±0.74 μM) are not significantly different. The k cat for ODNH2 (32.4±1.2 s−1) is twice as small as compared to the turnover numbers of the other substrates, viz. ODNHEtOH (61.6±5.0 s−1) and OD (54.4±2.0 s−1). The results suggest that N‐linoleoyl ethanolamide and N‐linoleoyl amide can be readily converted by lipoxygenases in vivo.

As yet, the physiological significance of hydroxylation of anandamide and linoleoyl amides is unknown. Therefore, we investigated whether hydroxylation of ODNHEtOH and ODNH 2 influences their binding abilities to the CB-1 receptor and whether it alters their reactivity towards a fatty acid amide hydrolase (FAAH) from rat brain. Neither the fatty acid amides nor their hydroxylated derivatives were able to displace the potent cannabinoid | 3 H]CP 55.940 from the CB-1 receptor (Äi> 1 u.M). Hydroxylation of ODNHEtOH resulted in a strong reduction of the maximum rate of hydrolysis by a FAAH, but the affinity of FAAH for the substrate remained of the same order of magnitude. Hydroxylation of ODNH 2 led to a decrease in the affinity of FAAH for the substrate, but its maximum rate of conversion was unaffected. Furthermore, hydroxylation of ODNHEtOH enhanced its capacity to inhibit competitively the hydrolysis of anandamide. The resulting prolonged lifetime of anandamide and other fatty acid amide derivatives may have a considerable impact on cellular signal transduction.