Among the biological activities of the endocannabinoid anandamide (N-arachidonoylethanolamine) (AEA), growing interest has been attracted by the regulation of mammalian fertility. Recently we have shown that treatment of mouse primary Sertoli cells with FSH enhances the activity of the AEA hydrolase [fatty acid amide hydrolase (FAAH)], though the molecular details were not elucidated. Here, we investigated whether FSH was also able to affect the enzymes that synthesize AEA (N-acyltransferase and N-acyl-phosphatidyl-ethanolamine-phospholipase D), the endogenous content of this endocannabinoid, and the level of the AEA-binding vanilloid receptor 1 (transient receptor potential channel vanilloid receptor subunit 1). We show that FSH enhanced FAAH activity (up to approximately 500% of the controls) and expression (up to approximately 300%), leading to a marked reduction (down to approximately 15%) of AEA content. However N-acyltransferase and N-acyl-phosphatidyl-ethanolamine-phospholipase D activity, and transient receptor potential channel vanilloid receptor subunit 1 binding were not affected. We also show that diacylglycerol lipase and monoacylglycerol lipase, which respectively synthesize and degrade 2-arachidonoyl-glycerol, were not regulated by FSH, neither was the membrane transport of this endocannabinoid. In addition, we show that FAAH stimulation by FSH was abrogated by inhibitors of protein kinase A (PKA) and cytochrome-P(450) aromatase, and was conversely mimicked by N,O'-dibutyryl cAMP and estrogen. Finally, we demonstrate that FSH protects Sertoli cells against the pro-apoptotic activity of AEA, through PKA and aromatase-dependent activation of FAAH. Altogether these data suggest that FAAH is the only target of FSH among the elements of the endocannabinoid system, and that its regulation by PKA and aromatase-dependent pathways impacts Sertoli cell proliferation.

The signaling activity of anandamide (AEA) is terminated by its uptake across the cellular membrane and subsequent intracellular hydrolysis by the fatty acid amide hydrolase (FAAH). To date, the existence of an AEA membrane transporter (AMT) independent of FAAH activity remains questionable, although it has been recently corroborated by pharmacological and genetic data. We performed confocal microscopy and biochemical analysis in human HaCaT keratinocytes, in order to study the cellular distribution of AMT and FAAH. We found that FAAH is intracellularly localized as a punctate staining partially overlapping with the endoplasmic reticulum. Consistently, subcellular fractionation and reconstitution of vesicles from membranes of different compartments demonstrated that FAAH activity was localized mainly in microsomal fractions, whereas AMT activity was almost exclusively in plasma membranes. These results provide the first morphological and biochemical evidence to support the view that transport and hydrolysis are two spatially and functionally distinct processes in AEA degradation.

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