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

Anandamide (AEA) has vasodilator activity, which can be terminated by cellular re-uptake and degradation. Here we investigated the presence and regulation of the AEA transporter in human umbelical vein endothelial cells (HUVECs). HUVECs take up AEA by facilitated transport (apparent K(m) = 190 +/- 10 nm and V(max) = 45 +/- 3 pmol. min(-1).mg(-1) protein), which is inhibited by alpha-linolenoyl-vanillyl-amide and N-(4-hydroxyphenyl)-arachidonoylamide, and stimulated up to 2.2-fold by nitric oxide (NO) donors. The NO scavenger hydroxocobalamin abolishes the latter effect, which is instead enhanced by superoxide anions but inhibited by superoxide dismutase and N-acetylcysteine, a precursor of glutathione synthesis. Peroxynitrite (ONOO(-)) causes a 4-fold activation of AEA transport into cells. The HUVEC AEA transporter contributes to the termination of a typical type 1 cannabinoid receptor (CB(1)) -mediated action of AEA, i.e. the inhibition of forskolin-stimulated adenylyl cyclase, because NO/ONOO(-) donors and alpha-linolenoyl-vanillyl-amide/N-(4-hydroxyphenyl)-arachidonoylamide were found to attenuate and enhance, respectively, this effect of AEA. Consistently, activation of CB(1) cannabinoid receptors by either AEA or the cannabinoid HU-210 caused a stimulation of HUVEC inducible NO synthase activity and expression up to 2.9- and 2. 6-fold, respectively. Also these effects are regulated by the AEA transporter. HU-210 enhanced AEA uptake by HUVECs in a fashion sensitive to the NO synthase inhibitor Nomega-nitro-l-arginine methyl ester. These findings suggest a NO-mediated regulatory loop between CB(1) cannabinoid receptors and AEA transporter

Two novel classes of neuroactive fatty acid amides are substrates for mouse neuroblastoma ‘anandamide amidohydrolase’

AbstractThe endogenous cannabimimetic substance, anandamide (N-arachidonoyl-ethanolamine) and the recently isolated sleep-inducing factor, oleoyl-amide (cis-9,10-octadecenoamide), belong to two neuroactive fatty acid amide classes whose action in mammals has been shown to be controlled by enzymatic amide bond hydrolysis. Here we report the partial characterisation and purification of ‘anandamide amidohydrolase’ from membrane fractions of N18 neuroblastoma cells, and provide evidence for a further and previously unsuspected role of this enzyme. An enzymatic activity catalysing the hydrolysis of [14C]anandamide was found in both microsomal and 10,000 × g pellet fractions. The latter fractions, which displayed the highest Vmax for anandamide, were used for further characterisation of the enzyme, and were found to catalyse the hydrolysis also of [14C]oleoyl-amide, with an apparent Km of 9.0 ± 2.2 μM. [14C]anandamide- and [14C]oleoyl-amide-hydrolysing activities: (i) exhibited identical pH- and temperature-dependency profiles; (ii) were inhibited by alkylating agents; (iii) were competitively inhibited by the phospholipase A2 inhibitor arachidonyl-trifluoromethyl-ketone with the same IC50 (3 μM); (iv) were competitively inhibited by both anandamide (or other polyunsaturated fatty acid-ethanolamides) and oleoyl-amide. Proteins solubilised from 10,000 × g pellets were directly analysed by isoelectric focusing, yielding purified fractions capable of catalysing the hydrolysis of both [14C]anandamide and [14C]oleoyl-amide. These data suggest that ‘anandamide amidohydrolase’ enzymes, such as that characterised in this study, may be used by neuronal cells also to hydrolyse the novel sleep-inducing factor oleoyl-amide

The Activity of Anandamide at Vanilloid VR1 Receptors Requires Facilitated Transport across the Cell Membrane and Is Limited by Intracellular Metabolism

The endogenous ligand of CB(1) cannabinoid receptors, anandamide, is also a full agonist at vanilloid VR1 receptors for capsaicin and resiniferatoxin, thereby causing an increase in cytosolic Ca(2+) concentration in human VR1-overexpressing (hVR1-HEK) cells. Two selective inhibitors of anandamide facilitated transport into cells, VDM11 and VDM13, and two inhibitors of anandamide enzymatic hydrolysis, phenylmethylsulfonyl fluoride and methylarachidonoyl fluorophosphonate, inhibited and enhanced, respectively, the VR1-mediated effect of anandamide, but not of resiniferatoxin or capsaicin. The nitric oxide donor, sodium nitroprusside, known to stimulate anandamide transport, enhanced anandamide effect on the cytosolic Ca(2+) concentration. Accordingly, hVR1-HEK cells contain an anandamide membrane transporter inhibited by VDM11 and VDM13 and activated by sodium nitroprusside, and an anandamide hydrolase activity sensitive to phenylmethylsulfonyl fluoride and methylarachidonoyl fluorophosphonate, and a fatty acid amide hydrolase transcript. These findings suggest the following. (i) Anandamide activates VR1 receptors by acting at an intracellular site. (ii) Degradation by fatty acid amide hydrolase limits anandamide activity on VR1; and (iii) the anandamide membrane transporter inhibitors can be used to distinguish between CB(1) or VR1 receptor-mediated actions of anandamide. By contrast, the CB(1) receptor antagonist SR141716A inhibited also the VR1-mediated effect of anandamide and capsaicin on cytosolic Ca(2+) concentration, although at concentrations higher than those required for CB(1) antagonism

N-oleoyldopamine, a novel endogenous capsaicin-like lipid that produces hyperalgesia.

N-Arachidonoyldopamine (NADA) was recently identified as an endogenous ligand for the vanilloid type 1 receptor (VR1). Further analysis of the bovine striatal extract from which NADA was isolated indicated the existence of substances corresponding in molecular mass to N-oleoyldopamine (OLDA), N-palmitoyldopamine (PALDA), and N-stearoyldopamine (STEARDA). Quadrupole time-of-flight mass spectrometric analysis of bovine striatal extracts revealed the existence of OLDA, PALDA, and STEARDA as endogenous compounds in the mammalian brain. PALDA and STEARDA failed to affect calcium influx in VR1-transfected human embryonic kidney (HEK) 293 cells or paw withdrawal latencies from a radiant heat source, and there was no evidence of spontaneous pain behavior. By contrast, OLDA induced calcium influx (EC(50) = 36 nm), reduced the latency of paw withdrawal from a radiant heat source in a dose-dependent manner (EC(50) = 0.72 microg), and produced nocifensive behavior. These effects were blocked by co-administration of the VR1 antagonist iodo-resiniferatoxin (10 nm for HEK cells and 1 microg/50 micro;l for pain behavior). These findings demonstrate the existence of an endogenous compound in the brain that is similar to capsaicin and NADA in its chemical structure and activity on VR1. Unlike NADA, OLDA was only a weak ligand for rat CB1 receptors; but like NADA, it was recognized by the anandamide membrane transporter while being a poor substrate for fatty-acid amide hydrolase. Analysis of the activity of six additional synthetic and potentially endogenous N-acyldopamine indicated the requirement of a long unsaturated fatty acid chain for an optimal functional interaction with VR1 receptors

Interactions between synthetic vanilloids and the endogenous cannabinoid system

Abstract The chemical similarity between some synthetic agonists of vanilloid receptors, such as olvanil (N-vanillyl-cis-9-octadecenoamide), and the`endocannabinoid' anandamide (arachidonoyl-ethanolamide, AEA), suggests possible interactions between the cannabinoid and vanilloid signalling systems. Here we report that olvanil is a stable and potent inhibitor of AEA facilitated transport into rat basophilic leukemia (RBL-2H3) cells. Olvanil blocked both the uptake and the hydrolysis of (ii) inhibited forskolin-induced cAMP formation in intact N18TG2 cells (IC SH = 1.60 W WM), this effect being reversed by the selective CB1 antagonist SR141716A. Pseudocapsaicin, but not capsaicin, also selectively bound to CB1 receptor-containing membranes. These data suggest that some of the analgesic actions of olvanil may be due to its interactions with the endogenous cannabinoid system, and may lead to the design of a novel class of cannabimimetics with potential therapeutic applications as analgesics. z 1998 Federation of European Biochemical Societies

Cloning of the first sn1-DAG lipases points to the spatial and temporal regulation of endocannabinoid signaling in the brain

Diacylglycerol (DAG) lipase activity is required for axonal growth during development and for retrograde synaptic signaling at mature synapses. This enzyme synthesizes the endocannabinoid 2-arachidonoyl-glycerol (2-AG), and the CB1 cannabinoid receptor is also required for the above responses. We now report on the cloning and enzymatic characterization of the first specific sn-1 DAG lipases. Two closely related genes have been identified and their expression in cells correlated with 2-AG biosynthesis and release. The expression of both enzymes changes from axonal tracts in the embryo to dendritic fields in the adult, and this correlates with the developmental change in requirement for 2-AG synthesis from the pre- to the postsynaptic compartment. This switch provides a possible explanation for a fundamental change in endocannabinoid function during brain development. Identification of these enzymes may offer new therapeutic opportunities for a wide range of disorders

Identification of a New Class of Molecules, the Arachidonyl Amino Acids, and Characterization of One Member That Inhibits Pain

In mammals, specific lipids and amino acids serve as crucial signaling molecules. In bacteria, conjugates of lipids and amino acids (referred to as lipoamino acids) have been identified and found to possess biological activity. Here, we report that mammals also produce lipoamino acids, specifically the arachidonyl amino acids. We show that the conjugate of arachidonic acid and glycine (N-arachidonylglycine (NAGly)) is present in bovine and rat brain as well as other tissues and that it suppresses tonic inflammatory pain. The biosynthesis of NAGly and its degradation by the enzyme fatty acid amide hydrolase can be observed in rat brain tissue. In addition to NAGly, bovine brain produces at least two other arachidonyl amino acids: N-arachidonyl gamma-aminobutyric acid (NAGABA) and N-arachidonylalanine. Like NAGly, NAGABA inhibits pain. These findings open the door to the identification of other members of this new class of biomolecules, which may be integral to pain regulation and a variety of functions in mammals

A novel fluorophosphonate inhibitor of the biosynthesis of the endocannabinoid 2-arachidonoylglycerol with potential anti-obesity effects

Background and Purpose The development of potent and selective inhibitors of the biosynthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG) via DAG lipases (DAGL) α and β is just starting to be considered as a novel and promising source of pharmaceuticals for the treatment of disorders that might benefit from a reduction in endocannabinoid tone, such as hyperphagia in obese subjects. Experimental Approach Three new fluorophosphonate compounds O-7458, O-7459 and O-7460 were synthesized and characterized in various enzymatic assays. The effects of O-7460 on high-fat diet intake were tested in mice. Key Results Of the new compounds, O-7460 exhibited the highest potency (IC50 = 690 nM) against the human recombinant DAGLα, and selectivity (IC50 > 10 μM) towards COS-7 cell and human monoacylglycerol lipase (MAGL), and rat brain fatty acid amide hydrolase. Competitive activity-based protein profiling confirmed that O-7460 inhibits mouse brain MAGL only at concentrations ≥10 μM, and showed that this compound has only one major ‘off-target’, that is, the serine hydrolase KIAA1363. O-7460 did not exhibit measurable affinity for human recombinant CB1 or CB2 cannabinoid receptors (Ki > 10 μM). In mouse neuroblastoma N18TG2 cells stimulated with ionomycin, O-7460 (10 μM) reduced 2-AG levels. When administered to mice, O-7460 dose-dependently (0–12 mg·kg−1, i.p.) inhibited the intake of a high-fat diet over a 14 h observation period, and, subsequently, slightly but significantly reduced body weight. Conclusions and Implications O-7460 might be considered a useful pharmacological tool to investigate further the role played by 2-AG both in vitro and in vivo under physiological as well as pathological conditions