Inhibition of Phospholipase Dα byN-Acylethanolamines

Abstract

N-Acylethanolamines (NAEs) are endogenous lipids in plants produced from the phospholipid precursor, N-acylphosphatidylethanolamine, by phospholipase D (PLD). Here, we show that seven types of plant NAEs differing in acyl chain length and degree of unsaturation were potent inhibitors of the well-characterized, plant-specific isoform of PLD-PLD␣. It is notable that PLD␣, unlike other PLD isoforms, has been shown not to catalyze the formation of NAEs from N-acylphosphatidylethanolamine. In general, inhibition of PLD␣ activity by NAEs increased with decreasing acyl chain length and decreasing degree of unsaturation, such that N-lauroylethanolamine and N-myristoylethanolamine were most potent with IC 50 s at submicromolar concentrations for the recombinant castor bean (Ricinus communis) PLD␣ expressed in Escherichia coli and for partially purified cabbage (Brassica oleracea) PLD␣. NAEs did not inhibit PLD from Streptomyces chromofuscus, and exhibited only moderate, mixed effects for two other recombinant plant PLD isoforms. Consistent with the inhibitory biochemical effects on PLD␣ in vitro, N-lauroylethanolamine, but not lauric acid, selectively inhibited abscisic acid-induced closure of stomata in epidermal peels of tobacco (Nicotiana tabacum cv Xanthi) and Commelina communis at low micromolar concentrations. Together, these results provide a new class of biochemical inhibitors to assist in the evaluation of PLD␣ physiological function(s), and they suggest a novel, lipid mediator role for endogenously produced NAEs in plant cells.Hydrolysis of membrane phospholipids by phospholipase D 2 (PLD, EC 3.1.4.4) activity in plants has been known for decades (Hanahan and Chaikoff, 1947); however, its precise physiological roles in plants are only beginning to be understood (for review, see Chapman et al., 1998;Munnik et al., 1998;Wang, 2001b). Recent evidence at the molecular level indicates that there are at least four functional isoforms of PLD in higher plants, designated ␣, ␤, ␥, and ␦, and their biochemical properties differ substantially (Wang, 2001a). PLD␣ catalyzes the wellcharacterized transphosphatidylation activity, which requires millimolar concentrations of Ca 2ϩ for optimal activity. At least two isoforms (␤ and ␥) appear to be optimally activated by micromolar concentrations of Ca 2ϩ and binding of inositol-containing phospholipids (Pappan et al., 1997), and these exhibit phospholipid substrate selectivity that differ markedly from that of PLD␣ (Pappan et al., 1998). The recently described PLD␦ activity is membrane associated and activated by free oleic acid .Evidence for the physiological function of PLD␣ points to a role in the degradation/reorganization of subcellular membranes, as well as a role in signal transduction (for review, see Chapman et al., 1998). This membrane degradation is manifested at the cellular level by loss of compartmentation leading to cell death, such as in phytohormone-initiated, PLDmediated senescence (Thompson, 1988;Fan et al., 1997). The unregulated activity of PLD␣ in plant cel...

SOBER1 phospholipase activity suppresses phosphatidic acid accumulation and plant immunity in response to bacterial effector AvrBsT

“…3C). We also tested N-acylethanolamine (NAE), a PLD␣-specific inhibitor (17). NAE did not alter the HR response, suggesting that the activity of other PLD isoforms likely contribute to PA production and HR activation.…”

N-Acylethanolamine Metabolism Interacts with Abscisic Acid Signaling inArabidopsis thalianaSeedlings

“…It was shown recently that increased PA levels in Arabidopsis lipid-phosphate phosphatase knockouts led to enhanced ABA sensitivity in germination tests (Katagiri et al, 2005). Since NAE12:0 has been shown previously to inhibit PLD-a activity (Austin-Brown and Chapman, 2002), it is possible that PA, which is a downstream product of PLD signaling, is elevated in FAAH overexpressors. The lower levels of endogenous NAEs in FAAH overexpressors could lead to elevated PLD-a activity and result in increased PA production and ABA hypersensitivity.…”

“…In this regard, it is worth noting that NAEs were found to be potent, noncompetitive inhibitors of PLD-a enzyme activity in vitro, with short-chain saturated species being most potent (effective concentrations in the nanomolar range). In fact, NAE12:0 was used by several groups to inhibit PLD-a-mediated processes in vivo (Austin-Brown and Chapman, 2002;Dhonukshe et al, 2003;Motes et al, 2005;Komis et al, 2006). In addition, NAE in mammals and ABA in plants also have been shown to exert their effects on various physiological processes through calcium and potassium channels, reactive oxygen species, nitric oxide release, Glu receptors, and sphingolipids (Coursol et al, 2003;Kang et al, 2004;Bright et al, 2006;Chai et al, 2006).…”

N-Acylated phospholipid metabolism and seedling growth

“…27,28 The potency of inhibition was inversely related to chain length and unsaturation, such that that shorter chain 12C saturated NAEs were more effective inhibitors that longer chain polyunsaturated species, but all types effectively reduced the activity of heterologously expressed Arabidopsis PLD-α1 toward common phospholipid (phosphatidylcholine, phosphatidylethanolamine) substrates. 27,29 These NAEs did not seem to have the same inhibitory effects on recombinant PLD-β/γ isoforms from Arabidopsis. However, at that time the complex family of PLD isoforms was incompletely characterized and it remains unknown if there are any effects by NAEs on the phospholipid-hydrolyzing activities of the other nine PLD isoforms.…”

“…Indeed, it was shown several years ago that various NAE types could function as inhibitors of PLD-α in vitro 27 and in vivo. 27,28 The potency of inhibition was inversely related to chain length and unsaturation, such that that shorter chain 12C saturated NAEs were more effective inhibitors that longer chain polyunsaturated species, but all types effectively reduced the activity of heterologously expressed Arabidopsis PLD-α1 toward common phospholipid (phosphatidylcholine, phosphatidylethanolamine) substrates. 27,29 These NAEs did not seem to have the same inhibitory effects on recombinant PLD-β/γ isoforms from Arabidopsis.…”