The ATP- and CoA-independent synthesis of arachidonoylethanolamide. A novel mechanism underlying the synthesis of the endogenous ligand of the cannabinoid receptor.

Recently, arachidonoylethanolamide was identified as the endogenous ligand of the delta 9 tetrahydrocannabinol receptor. Herein, we demonstrate that the synthesis of arachidonoylethanolamide is catalyzed by a novel CoA- and ATP-independent pathway, which is highly selective for arachidonic acid as the aliphatic constituent, is specific for ethanolamine as the polar moiety and occurs through a mechanism that utilizes a critical sulfhydryl residue. The production of arachidonoylethanolamide was calcium-independent and heat-labile and was selectively catalyzed by brain microsomal and cytosolic proteins among the tissues examined. Four independent lines of evidence demonstrate that the synthesis of arachidonoylethanolamide occurs through an ATP- and CoA-independent process. 1) The depletion of endogenous rabbit brain cytosolic or microsomal CoA and ATP by dialysis, or depletion of ATP by apyrase treatment, did not diminish the production of arachidonoylethanolamide; 2) the addition of exogenous ATP or CoA to cofactor depleted cytosol or microsomes did not stimulate arachidonoylethanolamide production; 3) the synthesis of arachidonoylethanolamide occurred in the absence of detectable arachidonoyl-CoA and did not correlate with either the amount of arachidonoyl-CoA in the incubation medium or the amount of arachidonoyl-CoA incorporated into polar and nonpolar lipids; 4) the addition of a 20-fold molar excess of unlabeled arachidonoyl-CoA to incubations containing [3H]arachidonic acid and ethanolamine did not attenuate the production of [3H]arachidonoylethanolamide by rabbit brain cytosolic or microsomal proteins. Collectively, these results demonstrate a novel chemical paradigm for the ATP- and CoA-independent conjugation of the carboxylate moiety of arachidonic acid to yield arachidonoylethanolamide, the first member of a new class of biologically active eicosanoid second messengers.

Rat and human pancreatic islet cells contain a calcium ion independent phospholipase A2 activity selective for hydrolysis of arachidonate which is stimulated by adenosine triphosphate and is specifically localized to islet beta-cells.

The recent demonstration that myocardial Ca(2+)-independent phospholipase A2 exists as a complex of catalytic and regulatory polypeptides that is modulated by ATP has suggested a novel mechanisms through which alterations in glycolytic flux can be coupled to the generation of eicosanoids which facilitate insulin secretion. To determine the potential relevance of this mechanism, we examined the kinetic characteristics, substrate specificities, and cellular locus of phospholipase A2 activity in pancreatic islets. Rat pancreatic islets contain a Ca(2+)-independent phospholipase A2 activity which is optimal at physiologic pH, preferentially hydrolyzes phospholipid substrates containing a vinyl ether linkage at the sn-1 position, and prefers arachidonic acid compared to oleic acid in the sn-2 position. Rat islet Ca(2+)-independent phospholipase A2 activity is inhibited by the mechanism-based inhibitor (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one and is stimulated by ATP. Purification of beta-cells from dispersed pancreatic islet cells by fluorescence-activated cell sorting demonstrated that beta-cells (but not non-beta-cells) contain Ca(2+)-independent, ATP-stimulated phospholipase A2 activity. Remarkably, clonal RIN-m5f insulinoma cells, which possess a defect in glucose-induced insulin secretion, contain a Ca(2+)-independent phospholipase A2 which is not modulated by alterations in ATP concentration. Collectively, these results and those of an accompanying paper [Ramanadham et al. (1993) Biochemistry (following paper in this issue)] implicate Ca(2+)-independent phospholipase A2 as a putative glucose sensor which can couple alterations in glycolytic metabolism to the generation of biologically active eicosanoids and thereby facilitate glucose-induced insulin secretion.

Calcium is sufficient but not necessary for activation of sheep platelet cytosolic phospholipase A2.

In this study we demonstrate that: (1) although the major phospholipase A2 present in sheep platelets is activated by calcium ions, it can effectively catalyze hydrolysis of the sn-2 ester linkage in phospholipids in the absence of calcium; (2) expression of calcium-independent phospholipase A2 activity can be induced by NaCl utilizing purified (but not crude) cytosolic enzyme; and (3) calcium-independent phospholipase A2 activity is regulated by a reconstitutable cytosolic protein. Collectively, these results underscore the fundamental catalytic differences between extracellular and intracellular calcium-dependent phospholipases A2 and demonstrate that calcium is sufficient, but not necessary, for the activation of this class of intracellular phospholipases A2.