<em>N</em>-Acylphosphatidylethanolamine Accumulation in Potato Cells upon Energy Shortage Caused by Anoxia or Respiratory Inhibitors^{<a href="#FN1" rid="FN1" class=" fn">1</a>}

Abstract

N-Acylphosphatidylethanolamine (NAPE) is an unusual phospholipid class that occurs in very small amounts (<1% of total phospholipids) in a wide range of organisms. Its main characteristic is the presence of a third fatty acyl residue linked to the N atom of the ethanolamine head group by an amide bond. The biochemical properties of NAPE and of its derivatives have been extensively covered in the comprehensive monograph of Schmid et al. (1990), whereas Chapman (2000) has reviewed the younger research carried out in plants.

In mammalian tissues, a characteristic feature of NAPE is its propensity to accumulate under various pathological conditions involving degenerative membrane changes. This occurs, for instance, in the infarcted aeras of dog myocardial tissue (Epps et al., 1979, 1980), in ischemic rat brain (Moesgaard et al., 1999, 2000), in Glu- (Hansen et al., 1998, 1999) or in sodium azide-induced (Hansen et al., 2000) neurotoxicity, after UV-B irradiation of mouse epidermal cells (Berdyshev et al., 2000), and in models of rat brain necrosis but not apoptosis (Hansen et al., 2001). Several roles have been attributed to NAPE, including membrane bilayer protection (Newman et al., 1986; Domingo et al., 1993; Hansen et al., 1999) and stabilization (Akoka et al., 1988; Domingo et al., 1994; Lafrance et al., 1997), as an endocannabinoid precursor involved in cell signaling processes (Schmid et al., 1996; Schmid, 2000), and finally in response to stress (Berdyshev et al., 2000).

Similar roles have been postulated for plant NAPE. In rehydrating cotton (Gossypium hirsutum) seeds, NAPE was suggested to act as a membrane-protecting and -stabilizing compound (Sandoval et al., 1995). The involvement of NAPE in elicitor-induced signaling in tobacco (Nicotiana tabacum) cells (Chapman et al., 1995a, 1998) and leaves (Tripathy et al., 1999) was suggested from the modulation by N-acylethanolamine of short-term (e.g. inhibition of elicitor-induced medium alkalinization) and long-term (e.g. induction of Phe ammonia lyase gene expression) defense responses (Tripathy et al., 1999). Increased NAPE synthesis was also observed in post-germinative seedlings submitted to chilling stress (Chapman and Sprinkle, 1996). Together, these data point to a general involvement of NAPE in all those processes where cellular structures are either challenged or severely compromised.

In the course of our studies on the effects of anoxic stress on cultured potato (Solanum tuberosum L. cv Bintje) cells, we have recently reported the occurrence of a threshold in energy production rate below which cells were irreversibly committed to death. Two phases thus were identified during anoxia treatment: a prelytic phase, during which cells still cope with the decreasing energy supply and remain intact, followed by an autolytic phase, characterized by an extensive hydrolysis of membrane lipids consecutive to the activation of a lipolytic acyl hydrolase (LAH; Rawyler et al., 1999). However, this major event is not the only change in lipid composition identified in these cells.

Here, we present the first evidence to our knowledge that anoxic stress of potato cells induces the formation of NAPE. This effect occurs correlatively to the LAH-dependent release of free fatty acids (FFAs) from membrane lipids, and is mimicked by the use of metabolic inhibitors under normoxia, pointing out energy shortage as the common origin of these lipid changes. We also show that the N-acyl pattern of NAPE molecules generated under anoxia is essentially insensitive to the changes that occur simultaneously in the FFA pool, whereas the corresponding O-acyl pattern differs markedly from that prevailing under normoxic conditions. The significance of NAPE formation in anoxic plant cells is discussed with special reference to its involvement in membrane protection and as a stress marker for cell death caused by the failure of metabolic homeostasis.

Labeled NAPE peaks of Figure ​Figure22 are described here by a group no. (Roman numerals) that refers to its total no. of acyl carbons and a lowercase letter associated to its own m/z value, and are listed as subgroups in the first column. The height of each labeled peak in Figure ​Figure22 was taken as a measure of its relative abundance. Groups were then made by summing the abundances of those species having the same number of acyl carbons. Note that in certain subgroups (II-c, II-d, III-a, III-b, III-c, and V-b), a single m/z value can yield more than one possible combination of acyl residues. Minor NAPE subgroups (II-a, II-d, II-f, III-d, and IV-a, accounting together for ≤10% of total NAPE) have been omitted for clarity.

Values are mean ± sd and the no. of replicates is given in italics in parentheses. The amount of diacyl lipids (represented by the membrane phospho- and glycolipids) is calculated by dividing the total fatty acid level (given on a molar basis) by a factor of 2.

Cell suspensions were aseptically fed with 2 mm each of NaN₃ and salicylhydroxamic acid (SHAM) to inhibit mitochondrial electron transport and then incubated for 0, 12, and 14 h under normoxia. After washing, cells were extracted and FFA and NAPE amounts determined as described in “Materials and Methods.” Values are mean ± sd and the no. of replicates is given in bracketed italics.

ACKNOWLEDGMENTS

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