Abstract
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 125I-labeled PLD), and does not cause gross damage to the neuronal membrane (as demonstrated by a lack of effect of PLD on [ 3H]γ-aminobutyric acid release). When neurons, labeled to isotopic equilibrium with [ 3H]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 [ 3H]arachidonate and [ 3H]diacylglycerol, respectively, but had no effect on NAE formation under these conditions. These experiments indicate that ∼ 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.
Highlights
Since the pioneering studies of Schmid and coworkers, it has been recognized that mammalian tissues have the ability to produce small quantities of saturated and unsaturated fatty acid ethanolamides (N-acylethanolamines, N-acyl ethanolamines (NAE))
In primary cultures of rat brain neurons, N-acyl phosphatidylethanolamine (NAPE) may serve as the precursor for anandamide, N-palmitoylethanolamine and other NAEs
The labeled neurons were washed with Dulbecco Modified Eagle's Medium (DMEM) and incubated for 10 min in D M E M containing one of the following phospholipases: phospholipase D (PLD), from Streptomyces chromofuscus (12.5 U/ml, Boehringer Mannheim or Sigma), phospholipase A2 (PLA2), from Apis mellifera (6.8 U/ml, Sigma) and phospholipase C (PLC), from Bacillus cereus (40 U/ml, Boehringer Mannheim)
Summary
Since the pioneering studies of Schmid and coworkers, it has been recognized that mammalian tissues have the ability to produce small quantities of saturated and unsaturated fatty acid ethanolamides (N-acylethanolamines, NAEs) (reviewed in Schmid et al, 1990). Two mechanisms have been proposed: the energy-independent condensation of ethanolamine and arachidonate, catalysed by anandamide synthetase activity (Deutsch and Chin, 1993; Devane and Axelrod, 1994; Kruszka and Gross, 1994); and the phospholipase D (PLD)-mediated cleavage of a membrane precursor phospholipid, N-acyl phosphatidylethanolamine (NAPE) (Di Marzo et al, 1994; Cadas et al, 1996) These two mechanisms need not be mutually exclusive (multiple paths of biosynthesis exist for many biologically active molecules, including several lipids) it would certainly be desirable to define their respective physiological roles in the formation of cannabimimetic NAEs. In primary cultures of rat brain neurons, NAPE may serve as the precursor for anandamide, N-palmitoylethanolamine and other NAEs. In primary cultures of rat brain neurons, NAPE may serve as the precursor for anandamide, N-palmitoylethanolamine and other NAEs Evidence supporting this possibility includes the existence of an enzymatic activity that catalyses the formation of NAEs from exogenous NAPE, as well as the occurrence of endogenous NAPE in unstimulated neurons (Di Marzo et al, 1994). We have investigated the cellular localization of NAPE in intact neurons in culture by probing their plasma membrane composition with exogenous phospholipases
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