Formation of Anandamide and Other N-Acylethanolamines

Before the discovery of anandamide, many aspects of the biogenesis of NAEs had been unraveled in a series of experiments carried out in the laboratory of Harald H.O. Schmid. These studies indicated that formation of NAEs likely proceeds from the hydrolytic cleavage of a phospholipid precursor, N-acylphos-phatidylethanolamine (NAPE), rather than from the condensation reaction of fatty acids with ethanolamine which had been suggested by earlier studies (ref. 32 and see below). Our own work on primary cultures of rat brain neurons corroborated and extended the pathway predicted by Schmid and coworkers (Fig. 6.10). 

Primary cultures of neurons, but not astrocytes, contain detectable quantities of a lipid component which we have identified as NAPE by enzymatic cleavage, multiple chromatographic analyses and nuclear magnetic resonance spectroscopy. Neuronal NAPE is composed of a variety of molecular species, which differ in the fatty acyl group bound, through an amide bond, to the ethanolamine moiety of phosphatidylethanolamine (PE). We have found at least five such molecular species in cultured neurons (Table 6.1). 

How is NAPE synthesized, and how is its biosynthesis controlled Unfortunately, we cannot satisfactorily answer this question at present. A calcium-dependent enzyme activity that catalyzes the transfer of fatty acyl groups from various donor phospholipids to PE, producing NAPE, has been partially characterized in dog brain tissue and in primary cultures of rat brain neurons.In these neurons, NAPE biosynthesis is stimulated by stimuli that elevate intracellular calcium levels and is potentiated by cAMP-dependent protein kinase activity. But the physiological roles, anatomical distribution and regulation of NAPE biosynthesis in the adult nervous system remain unknown. 

In what cellular compartment is NAPE localized Again, we can provide only a very partial answer to this question. Experiments in which the membranes of intact neurons were probed with bacterial PLD Streptomyces chromofuscus) suggest that a large portion of NAPE (at least 40%) is collected in the plasma membrane, where one would expect a precursor for an intercellular signaling molecule.  

Hydrolytic cleavage of membrane NAPE by a stimulus-activated PLD activity yields, in a single-step reaction, saturated and unsaturated NAEs. In fact, the NAEs recovered after stimulation of neurons in primary culture closely correspond to those expected from the structure of neuronal NAPE. This is an important piece of evidence in favor of a role of NAPE as NAE precursor but not the only one. Other supportive evidences include the ability of homogenates of neurons and other tissues (e.g., testis) to hydrolyze radiolabeled NAPE forming NAEs and the demonstration that NAPE turnover accompanies NAE formation in stimulated neurons. 

---