N-Acylethanolamines Are Metabolized by Lipoxygenase and Amidohydrolase in Competing Pathways during Cottonseed Imbibition

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...

Molecular Identification of a Functional Homologue of the Mammalian Fatty Acid Amide Hydrolase in Arabidopsis thaliana

“…Hydrolysis of NAEs was reconstituted and characterized in microsomes of cottonseeds and appeared to be catalyzed by an enzyme similar to the FAAH of mammalian species (18). Here we report the identification of a plant orthologue of mammalian FAAH by bioinformatic approaches, isolation of its cDNA sequence, expression of this cDNA in Escherichia coli, and identification of the protein product as an NAE amidohydrolase.…”

“…Recently, depletion of NAEs during seed imbibition/germination was determined to occur via two metabolic pathways: one lipoxygenase-mediated for the formation of NAE oxylipins from NAE 18:2, and one amidase-mediated for hydrolysis of saturated and unsaturated NAEs (18). Hydrolysis of NAEs was reconstituted and characterized in microsomes of cottonseeds and appeared to be catalyzed by an enzyme similar to the FAAH of mammalian species (18).…”

N-Acylethanolamine Signaling in Tobacco Is Mediated by a Membrane-Associated, High-Affinity Binding Protein

“…Curiously NAE 18:0, NAE 18:1, and NAE 18:3 were active, whereas NAE 18:2 was not. The reason for this difference in PAL induction is unclear at this point, but may reflect a different fate for NAE 18:2, because this NAE was shown to be metabolized by lipoxygenase-13 pathway in germinating seeds (Shrestha et al, 2002). Thus, although NAE modulation of short and long-term defense responses in plants shares some similarities with the endocannabinoid-signaling pathways in animals, there are some distinct differences with respect to NAE types.…”

“…3) nor affected [ 3 H]NAE 14:0-specific binding in microsomal membranes (Fig. 5A), although this may be a consequence of its rapid peroxidation by microsomal membranes (Shrestha et al, 2002), because DDM-solubilized NAE-binding activity appears to interact with NAE18:2 (Fig. 5B).…”

“…5A), suggesting that NAE 18:2 does bind to the putative NAE receptor. As mentioned above, NAE 18:2 is readily converted by a microsomal 13-LOX to novel NAE oxylipins (Shrestha et al, 2002) that may not interact with the NAE-binding protein. This functional NAE-LOX activity, although present in microsomes, may not be present in our DDM-solubilized preparation, and consequently, unmetabolized NAE18:2 has access to the NAE-binding protein.…”