Membrane localization of N-acylphosphatidylethanolamine in central neurons: Studies with exogenous phospholipases

We studied the localization of N-acyl phosphatidylethanolamine (NAPE), a putative cannabinoid precursor, in primary cultures of striatal and cortical neurons from the rat brain. We probed intact neurons with various exogenous phospholipases, including S. chromofuscus phospholipase D (PLD). S. chromofuscus PLD does not penetrate into neurons (as demonstrated by a lack of internalization of I-125-labeled PLD), and does not cause gross damage to the neuronal membrane (as demonstrated by a lack of effect of PLD on [H-3]gamma-aminobutyric acid release). When neurons, labeled to isotopic equilibrium with [H-3]ethanolamine, were incubated for 10 min with S. chromofuscus PLD, approximately 50% of neuronal NAPE was hydrolysed. This hydrolysis was accompanied by the release of a family of N-acyl ethanolamines (NAE) (assessed by high performance liquid chromatography), which included the cannabinoid receptor agonist, anandamide. Exogenous phospholipase A(2) (PLA(2)) (Apis mellifera) and PLC (B. cereus) mobilized [H-3]arachidonate and [H-3]diacylglycerol, respectively, but had no effect on NAE formation under these conditions. These experiments indicate that approximate to 50% of neuronal NAPE is localized in a compartment that is easily accessible to extracellular PLD, possibly the plasmalemma, where it would also be easily hydrolyzed upon stimulation to produce NAE.