anandamide and Leukemia--Basophilic--Acute

The uptake of arachidonoyl ethanolamide (anandamide, AEA) in rat basophilic leukemia cells (RBL-2H3) has been proposed to occur via a saturable transporter that is blocked by specific inhibitors. Measuring uptake at 25 s, when fatty acid amide hydrolase (FAAH) does not appreciably affect uptake, AEA accumulated via a nonsaturable mechanism at 37 degrees C. Interestingly, saturation was observed when uptake was plotted using unbound AEA at 37 degrees C. Such apparent saturation can be explained by rate-limited delivery of AEA through an unstirred water layer surrounding the cells (1). In support of this, we observed kinetics consistent with rate-limited diffusion at 0 degrees C. Novel transport inhibitors have been synthesized that are either weak FAAH inhibitors or do not inhibit FAAH in vitro (e.g. UCM707, OMDM2, and AM1172). In the current study, none of these purported AEA transporter inhibitors affected uptake at 25 s. Longer incubation times illuminate downstream events that drive AEA uptake. Unlike the situation at 25 s, the efficacy of these inhibitors was unmasked at 5 min with appreciable inhibition of AEA accumulation correlating with partial inhibition of AEA hydrolysis. The uptake and hydrolysis profiles observed with UCM707, VDM11, OMDM2, and AM1172 mirrored two selective and potent FAAH inhibitors CAY10400 and URB597 (at low concentrations), indicating that weak inhibition of FAAH can have a pronounced effect upon AEA uptake. At 5 min, the putative transport inhibitors did not reduce AEA uptake in FAAH chemical knock-out cells. This strongly suggests that the target of UCM707, VDM11, OMDM2, and AM1172 is not a transporter at the plasma membrane but rather FAAH, or an uncharacterized intracellular component that delivers AEA to FAAH. This system is therefore unique among neuro/immune modulators because AEA, an uncharged hydrophobic molecule, diffuses into cells and partial inhibition of FAAH has a pronounced effect upon its uptake.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Calcium Channel Blockers; Cell Culture Techniques; Cell Membrane; Diffusion; Endocannabinoids; Humans; Hydrolysis; Kinetics; Leukemia, Basophilic, Acute; Polyunsaturated Alkamides; Rats; Serum Albumin, Bovine

Indirect evidence for the existence of a specific protein-mediated process for the cellular uptake of endocannabinoids has been reported, but recent results suggested that such a process, at least for AEA [ N -arachidonoylethanolamine (anandamide)], is facilitated uniquely by its intracellular hydrolysis by FAAH (fatty acid amide hydrolase) [Glaser, Abumrad, Fatade, Kaczocha, Studholme and Deutsch (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 4269-4274]. In the present study, we show that FAAH alone cannot account for the facilitated diffusion of AEA across the cell membrane. In particular, (i) using a short incubation time (90 s) to avoid AEA hydrolysis by FAAH, AEA accumulation into rat basophilic leukaemia or C6 cells was saturable at low microM concentrations of substrate and non-saturable at higher concentrations; (ii) time-dependent and, at low microM concentrations of substrate, saturable AEA accumulation was observed also using mouse brain synaptosomes; (iii) using synaptosomes prepared from FAAH-deficient mice, saturable AEA accumulation was still observed, although with a lower efficacy; (iv) when 36 AEA and N -oleoylethanolamine analogues, most of which with phenyl rings in the polar head group region, were tested as inhibitors of AEA cellular uptake, strict structural and stereochemical requirements were needed to observe significant inhibition, and in no case the inhibition of FAAH overlapped with the inhibition of AEA uptake; and (v) AEA biosynthesis by cells and sensory neurons was followed by AEA release, and this latter process, which cannot be facilitated by FAAH, was still blocked by an inhibitor of AEA uptake. We suggest that at least one protein different from FAAH is required to facilitate AEA transport across the plasma membrane in a selective and bi-directional way.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Biological Transport; Brain Chemistry; Carrier Proteins; Cell Line, Tumor; Cell Membrane; Cells, Cultured; Endocannabinoids; Enzyme Inhibitors; Ganglia, Spinal; Humans; Kidney; Leukemia, Basophilic, Acute; Male; Mice; Mice, Knockout; Neoplasm Proteins; Organophosphonates; Polyunsaturated Alkamides; Rats; Synaptosomes; Thapsigargin

The endogenous cannabinoid receptor agonist anandamide (AEA) and the related compound palmitoylethanolamide (PEA) are inactivated by transport into cells followed by metabolism by fatty acid amide hydrolase (FAAH). The cellular uptake of AEA has been characterized in detail, whereas less is known about the properties of the PEA uptake, in particular in neuronal cells. In the present study, the pharmacological and functional properties of PEA and AEA uptake have been investigated in mouse Neuro-2a neuroblastoma and, for comparison, in rat RBL-2H3 basophilic leukaemia cells. Saturable uptake of PEA and AEA into both cell lines were demonstrated with apparent K(M) values of 28 microM (PEA) and 10 microM (AEA) in Neuro-2a cells, and 30 microM (PEA) and 9.3 microM (AEA) in RBL-2H3 cells. Both PEA and AEA uptake showed temperature-dependence but only the AEA uptake was sensitive to treatment with Pronase and phenylmethylsulfonyl fluoride. The AEA uptake was inhibited by AM404, 2-arachidonoylglycerol (2-AG), R1- and S1-methanandamide, arachidonic acid and olvanil with similar potencies for the two cell types. PEA, up to a concentration of 100 microM, did not affect AEA uptake in either cell line. AEA, 2-AG, arachidonic acid, R1-methanandamide, (9)-THC, and cannabidiol inhibited PEA transport in both cell lines. The non-steroidal anti-inflammatory drug indomethacin inhibited the AEA uptake but had very weak effects on the uptake of PEA. From these data, it can be concluded that PEA is transported in to cells both by passive diffusion and by a facilitated transport that is pharmacologically distinguishable from AEA uptake.

Topics: Amides; Animals; Arachidonic Acids; Brain Neoplasms; Cannabinoids; Cell Survival; Endocannabinoids; Ethanol; Ethanolamines; Kinetics; Leukemia, Basophilic, Acute; Mast Cells; Mice; Neuroblastoma; Palmitic Acids; Phenylmethylsulfonyl Fluoride; Polyunsaturated Alkamides; Pronase; Protease Inhibitors; Rats; Tumor Cells, Cultured

Fatty acid amide hydrolase (FAAH) catalyzes the hydrolysis of bioactive fatty acid amides and esters such as the endogenous cannabinoid receptor ligands, anandamide (N-arachidonoyl-ethanolamine) and 2-arachidonoylglycerol, and the putative sleep inducing factor cis-9-octadecenoamide (oleamide). Most FAAH blockers developed to date also inhibit cytosolic phospholipase A2 (cPLA2) and/or bind to the CB1 cannabinoid receptor subtype. Here we report the finding of four novel FAAH inhibitors, two of which, malhamensilipin A and grenadadiene, were screened out of a series of thirty-two different algal natural products, and two others, arachidonoylethylene glycol (AEG) and arachidonoyl-serotonin (AA-5-HT) were selected out of five artificially functionalized polyunsaturated fatty acids. When using FAAH preparations from mouse neuroblastoma N18TG2 cells and [14C]anandamide as a substrate, the IC50s for these compounds ranged from 12.0 to 26 microM, the most active compound being AA-5-HT. This substance was also active on FAAH from rat basophilic leukaemia (RBL-2H3) cells (IC50 = 5.6 microM), and inhibited [14C]anandamide hydrolysis by both N18TG2 and RBL-2H3 intact cells without affecting [14C]anandamide uptake. While AEG behaved as a competitive inhibitor and was hydrolyzed to arachidonic acid (AA) by FAAH preparations, AA-5-HT was resistant to FAAH-catalyzed hydrolysis and behaved as a tight-binding, albeit non-covalent, mixed inhibitor. AA-5-HT did not interfere with cPLA2-mediated, ionomycin or antigen-induced release of [3H]AA from RBL-2H3 cells, nor with cPLA2 activity in cell-free experiments. Finally, AA-5-HT did not activate CB1 cannabinoid receptors since it acted as a very weak ligand in in vitro binding assays, and, at 10-15 mg/kg body weight, it was not active in the 'open field', 'hot plate' and rectal hypothermia tests carried out in mice. Conversely AEG behaved as a cannabimimetic substance in these tests as well as in the 'ring' immobility test where AA-5-HT was also active. AA-5-HT is the first FAAH inhibitor reported to date which is inactive both against cPLA2 and at CB1 receptors, whereas AEG represents a new type of cannabinoid receptor agonist.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Cyclopropanes; Endocannabinoids; Enzyme Inhibitors; Esters; Ethylene Glycols; Kinetics; Leukemia, Basophilic, Acute; Lipids; Mice; Neuroblastoma; Polyunsaturated Alkamides; Rats; Serotonin; Structure-Activity Relationship; Substrate Specificity; Tumor Cells, Cultured

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 [14C]AEA by intact RBL-2H3 cells (IC50 = 9 microM), while capsaicin and pseudocapsaicin (N-vanillyl-nonanamide) were much less active. Olvanil was more potent than previously reported inhibitors of AEA facilitated transport, i.e. phloretin (IC50 = 80 microM), AM404 (12.9% inhibition at 10 microM) or oleoylethanolamide (27.5% inhibition at 10 microM). Olvanil was a poor inhibitor of [14C]AEA hydrolysis by RBL-2H3 and N18TG2 cell membranes, suggesting that the inhibitory effect on [14C]AEA breakdown observed in intact cells was due to inhibition of [14C]AEA uptake. Olvanil was stable to enzymatic hydrolysis, and (i) displaced the binding of high affinity cannabinoid receptor ligands to membrane preparations from N18TG2 cells and guinea pig forebrain (Ki = 1.64-7.08 microM), but not from cells expressing the CB2 cannabinoid receptor subtype; (ii) inhibited forskolin-induced cAMP formation in intact N18TG2 cells (IC50 = 1.60 microM), 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.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Arachidonic Acids; Cannabinoid Receptor Modulators; Cannabinoids; Capsaicin; Cell Line; Endocannabinoids; Kinetics; Leukemia, Basophilic, Acute; Macrophages; Mice; Neuroblastoma; Polyunsaturated Alkamides; Rats; Receptors, Drug; Tumor Cells, Cultured