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

Rhidaya Shrestha , Minke A. Noordermeer , Marcelis Van der Stelt , Gerrit A. Veldink , and Kent D. Chapman ^*

Department of Biological Sciences, Division of Biochemistry and Molecular Biology, University of North Texas, Denton, Texas 76203 (R.S., K.D.C.); and Bijvoet Center for Biomolecular Research, Department of Bio-organic Chemistry, Utrecht University, The Netherlands (M.A.N., M.V.d.S., G.A.V.)

^* Corresponding author; email: chapman{at}unt.edu.

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 [1-¹⁴C]N-palmitoylethanolamine was used as a substrate, free fatty acids (FFA) were produced by extracts of imbibed cottonseeds. When synthetic [1-¹⁴C]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.

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