Neurochem. 69, 631 -638 (1997).N-Arachidonoylethanolamine (AEA; also called anandamide) has been isolated from brain and shown to bind and activate the brain cannabinoid receptor (Devane et al., 1992). Exogenously administered AEA mimics many of the effects of z9-tetrahydrocannabinol (z9-THC), including the production of hypothermia, decreased spontaneous activity and analgesia in rodents (Fride and Mechoulam, 1993), inhibition of adenylyl cyclase (Vogel et al., 1993), and decreased opening of voltage-operated calcium channels (Mache et al., 1993). Recent studies have demonstrated that AEA is released from neurons in primary culture in response to increased intracellular calcium (DiMarzo et al., 1994), which supports the hypothesis that AEA functions in the brain as a signaling molecule.AEA is rapidly catabolized by brain membranes to arachidonic acid and ethanolamine through the action of an amidohydrolase that is selective for fatty acyl amides (Deutsch and Chin, 1993;Desarnaud et al., 1995;Hillard et al., 1995a;Cravatt et al., 1996). Although AEA amidohydrolase activity is high in crude membrane preparations from brain, it is quite low in tissue preparations that are enriched in plasma membranes (Hillard et al., 1995a). These results suggest that AEA released into the extracellular space is not inactivated by hydrolysis to arachidonic acid. The present experiments explore an alternative mechanism for AEA inactivation, specifically, cellular reuptake of AEA. DiMarzo and coworkers (1994) have reported that cortical neurons in primary culture accumulate AEA in a saturable manner. These investigators further reported that AEA accumulationwas rapid, was temperature sensitive, and was not inhibited by either arachidonic acid or N-palmitoylethanolamine. With the studies reported here, we describe and characterize a similar transport process for AEA in cerebellar granule cells. AEA transport is saturable, temperature dependent, specific, and inhibited by phloretin. The uptake process has the characteristics of facilitated diffusion, that is, it is reversible and not dependent on cellular ATP. These results suggest, first, that AEA distribution between intracellular and extracellular compartments in cerebellar granule cells is dependent on facilitated diffusion and, second, that AEA permeability across

A number of endothelium-derived relaxing factors have been identified including nitric oxide, prostacyclin, and the epoxyeicosatrienoic acids. Previous work showed that in rabbit aortic endothelial cells, arachidonic acid was metabolized by a lipoxygenase to vasodilatory eicosanoids. The identity was determined by the present study. Aortic homogenates were incubated in the presence of [U-14 C]arachidonic acid, [U-14 C]arachidonic acid plus 15-lipoxygenase (soybean lipoxidase), or [U-14 C]15-hydroxyeicosatetraenoic acid (15-HPETE) and analyzed by reverse phase high pressure liquid chromatography (RP-HPLC). Under both experimental conditions, there was a radioactive metabolite that migrated at 17.5-18.5 min on RP-HPLC. When the metabolite was isolated from aortic homogenates, it relaxed precontracted aortas in a concentration-dependent manner. Gas chromatography/mass spectrometry (GC/MS) of the derivatized metabolite indicated the presence of two products; 11,12,15-trihydroxyeicosatrienoic acid (THETA) and 11,14,15-THETA. A variety of chemical modifications of the metabolite supported these structures and confirmed the presence of a carboxyl group, double bonds, and hydroxyl groups. With the combination of 15-lipoxygenase, arachidonic acid, and aortic homogenate, an additional major radioactive peak was observed. This fraction was analyzed by GC/ MS. The mass spectrum was consistent with this peak, containing both the 11-hydroxy-14,15-epoxyeicosatrienoic acid (11-H-14,15-EETA) and 15-H-11,12-EETA. The hydroxyepoxyeicosatrienoic acid (HEETA) fraction also relaxed precontracted rabbit aorta. Microsomes derived from rabbit aortas also synthesized 11,12,15-and 11,14,15-THETAs from 15-HPETE, and pretreatment with the cyctochrome P450 inhibitor, miconazole, blocked the formation of these products. The present studies suggest that arachidonic acid is metabolized by 15-lipoxygenase to 15-HPETE, which undergoes an enzymatic rearrangement to 11-H-14,15-EETA and 15-H-11,12-EETA. Hydrolysis of the epoxy group results in the formation of 11,14,15-and 11,12,15-THETA, which relaxed rabbit aorta. Thus, the 15-series THETAs join prostacyclin, nitric oxide, and epoxyeicosatrienoic acids as new members of the family of endothelium-derived relaxing factors.The vascular endothelium synthesizes and releases compounds that are involved in the regulation of vascular tone (1). These endothelial-derived vasoactive compounds include prostacyclin, endothelium-derived relaxing factor or nitric oxide, endothelium-derived hyperpolarizing factor, endothelium-derived contracting factor, and endothelin. These endothelial factors mediate the vasoactive effects of a number of hormones including acetylcholine, bradykinin, and ATP (1). Alterations in the production of these compounds may be associated with cardiovascular diseases, including atherosclerosis, coronary vasospasm, and hypertension.Arachidonic acid is metabolized by the vascular endothelium to a variety of cyclooxygenase, lipoxygenase, and cytochrome P450 epoxygenase products (2). The iden...

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It has been reported that the endogenous cannabinoid N-arachidonylethanolamide (AEA), commonly referred to as anandamide, has the characteristics of an endothelium-derived hyperpolarizing factor in rat mesenteric artery. We have carried out studies to determine whether AEA affects coronary vascular tone. The vasorelaxant effects of AEA were determined in isolated bovine coronary artery rings precontracted with U-46619 (3 × 10−9 M). AEA decreased isometric tension, producing a maximal relaxation of 51 ± 9% at a concentration of 10−5 M. Endothelium-denuded coronary arteries were not significantly affected by AEA. The CB1 receptor antagonist SR-141716A (10−6 M) failed to reduce the vasodilatory effects of AEA, suggesting that the CB1 receptor is not involved in this action of AEA. Because AEA is rapidly converted to arachidonic acid and ethanolamine in brain and liver by a fatty acid amide hydrolase (FAAH), we hypothesized that the vasodilatory effect of AEA results from its hydrolysis to arachidonic acid followed by enzymatic conversion to vasodilatory eicosanoids. In support of this hypothesis, bovine coronary arteries incubated with [3H]AEA for 30 min hydrolyzed 15% of added substrate; ∼9% of the radiolabeled product was free arachidonic acid, and 6% comigrated with the prostaglandins (PGs) and epoxyeicosatrienoic acids (EETs). A similar result was obtained in cultured bovine coronary endothelial cells. Inhibition of the FAAH with diazomethylarachidonyl ketone blocked both the metabolism of [3H]AEA and the relaxations to AEA. Whole vessel and cultured endothelial cells prelabeled with [3H]arachidonic acid synthesized [3H]PGs and [3H]EETs, but not [3H]AEA, in response to A-23187. Furthermore, SR-141716A attenuated A-23187-stimulated release of [3H]arachidonic acid, suggesting that it may have actions other than inhibition of CB1 receptor. These experiments suggest that AEA produces endothelium-dependent vasorelaxation as a result of its catabolism to arachidonic acid followed by conversion to vasodilatory eicosanoids such as prostacyclin or the EETs.

Two putative endocannabinoids, N-arachidonylethanolamine (AEA) and 2-arachidonylglycerol, are inactivated by removal from the extracellular environment by a process that has the features of protein-mediated facilitated diffusion. We have synthesized and studied 22 N-linked analogues of arachidonylamide for the purpose of increasing our understanding of the structural requirements for the binding of ligands to the AEA transporter. We have also determined the affinities of these analogues for both the CB 1 cannabinoid receptor and fatty acid amide hydrolase (FAAH). We have identified several structural features that enhance binding to the AEA transporter in cerebellar granule cells. We have confirmed the findings of others that replacing the ethanolamine head group with 4-hydroxybenzyl results in a high-affinity ligand for the transporter. However, we find that the same molecule is also a competitive inhibitor of FAAH. Similarly, replacement of the ethanolamine of AEA with 3-pyridinyl also results in a high-affinity inhibitor of both the transporter and FAAH. We conclude that the structural requirements for ligand binding to the CB 1 receptor and binding to the transporter are very different; however, the transporter and FAAH share most, but not all, structural requirements. Key Words: N-ArachidonylethanolamineEndocannabinoid-Fatty acid amide hydrolase-Cannabinoid receptor.