Anandamide Uptake by Human Endothelial Cells and Its Regulation by Nitric Oxide

Abstract

Anandamide (arachidonoylethanolamide, AEA) 1 belongs to an emerging class of endogenous lipids including amides and esters of long chain polyunsaturated fatty acids and is collectively termed "endocannabinoids" (1, 2). In fact, AEA has been isolated and characterized as an endogenous ligand for both CB 1 and, to a lesser extent, CB 2 cannabinoid receptor subtypes, and has been shown to mimic the psychotropic, antiemetic, and analgesic effects of cannabinoids (3). Recently, attention has been focused on the cardiovascular actions of AEA and their potential role in human shock conditions (4). In particular, a role for AEA has been proposed in both endothelium-dependent and -independent relaxations of vascular tissues, which involve several mechanisms including hyperpolarization of the smooth muscle cell membrane (5-7). A similar mechanism has been attributed in the past to a diffusible endothelium-derived hyperpolarizing factor (EDHF) different from nitric oxide (NO), whose chemical nature is still a matter of speculation (8). In fact, AEA has been proposed as an EDHF (5), though this hypothesis is still under debate (9, 10), and recent data strongly support the theory that EDHF is a cytochrome P450 metabolite (11). Whether or not an EDHF, AEA is likely to play an important role in the control of vascular tone (for reviews see Refs. 4 and 12), as suggested also by the observation that both endothelial cells and macrophages release this as well as the other endocannabinoid, 2-arachidonoyl-glycerol (2-AG) (1, 13-16).The pharmacological effects of AEA on CB 1 and CB 2 receptors depend, as for any other extracellular transmitter, on its life span in the extracellular space, which is limited by a two-step process: (i) its rapid and selective uptake by cells through the action of a membrane transporter and (ii) intracellular degradation. In particular, AEA is hydrolyzed to ethanolamine and arachidonic acid by the enzyme fatty acid amide hydrolase (FAAH) (17,18). Both components of this inactivation process of AEA are the objects of active investigations. Recent data seem to indicate that the uptake process is the rate-limiting step in AEA degradation (19 -23). There is pharmacological evidence suggesting that also the hypotensive action of AEA in vivo is limited by its re-uptake (24). However, the existence of the AEA membrane transporter in endothelial cells has never been investigated.Although cannabinoid receptor activation was recently shown to lead to AEA biosynthesis (25,26), the possibility of a functional link between CB 1 and CB 2 receptors and the AEA transporter has not been tested. Such a functional coupling might trigger self-elimination of AEA following activation by this lipid of cannabinoid receptor-dependent signaling pathways and would represent a regulatory loop critical for the manifold actions of this compound. A possible mechanism for this coupling may be suggested by findings that AEA binding to

Retrograde endocannabinoid signaling at striatal synapses requires a regulated postsynaptic release step

“…GABA A receptors also are not obligatory because we observed depression of EPSCs when picrotoxin was included in the aCSF (2). NO has been reported to regulate AMT activity (40), but treatments that block NOS or scavenge NO did not alter AEA-induced depression, indicating that this messenger is not involved.…”

“…2b), showing that L-type calcium channels are not involved at a stage when eCB levels are already high. Nitric oxide (NO) has been reported to enhance the activity of the AMT (16,(38)(39)(40). However, neither inhibition of NO production by 100 M N -Nitro-L-arginine methyl ester hydrochloride (L-NAME) nor 500 mg/liter hemoglobin, the NO scavenger, was able to block AEA-induced depression [EPSC amplitude ϭ 66 Ϯ 7.9%; n ϭ 7; P Ͻ 0.001 (L-NAME); 66 Ϯ 11%; n ϭ 4; P Ͻ 0.05 (hemoglobin)] (Fig.…”

Anandamide Induces Apoptosis in Human Cells via Vanilloid Receptors

“…In C6 or DAUDI cells, the effects on PCD of co-administration of the transporter inhibitor AM404, which increases extracellular concentration of AEA, or of CBR antagonists SR141716 and SR144528, which prevent CBR activation (Table III), support this concept. These findings can be interpreted by suggesting a regulatory loop between CB receptors and the AEA transporter, which has been recently demonstrated in human endothelial cells (13). In this loop, the binding of AEA to CB receptors triggers the activation of AEA uptake by cells, followed by intracellular degradation of AEA by FAAH.…”

Inhibition of endocannabinoid metabolism attenuates enhanced hippocampal neuronal activity induced by kainic acid

“…AEA and 2-AG are synthesized upon demand and released from postsynaptic sites to act at presynaptic CB1 receptors, dampening inhibitory input from basket cells to glutamatergic pyramidal cells (Wilson and Nicoll, 2001). AEA and 2-AG are removed from the extracellular synaptic space by a putative reuptake system (Deutsch and Chin, 1993;Hillard et al, 1997;Maccarrone et al, 2000;Piomelli et al, 1999) and are metabolized by fatty acid amide hydrolase (FAAH) and monoacylglycerol (MAG) lipase, respectively (Cravatt et al, 1996;Cravatt and Lichtman, 2003;Hillard et al, 1995).…”