Anandamide Hydrolysis by Human Cells in Culture and Brain

Maccarrone, Mauro; Stelt, Marcelis van der; Rossi, Antonello; Veldink, Gerrit A.; Vliegenthart, Johannes F.G.; Agró, Alessandro Finazzi

doi:10.1074/jbc.273.48.32332

Cited by 182 publications

(227 citation statements)

References 44 publications

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“…The K M values estimated for the facilitated transport component of AEA are rather similar to those found previously (Hillard et al, 1997;Bisogno et al, 1997;Rakhshan et al, 2000), although other authors have reported K M values near or below 1 mM Maccarrone et al, 1998;2000b). One explanation for this discrepancy may be di erences in curve ®tting techniques (see Rakhshan et al, 2000), and it can be noted that in a recent study using human CCF-STTG1 astrocytoma cells, the IC 50 of non-radioactive AEA as an inhibitor of the uptake of 30 nM AEA (15.1+3.0 mM) was considerably higher than its K M value (0.6+0.1 mM) .…”

Section: Discussionsupporting

confidence: 89%

“…Once again, this would suggest that the contribution of FAAH to the uptake process is of pharmacological importance. This may also explain the di erences in sensitivity to arachidonic acid found in the di erent laboratories, given that it is an FAAH inhibitor (Maccarrone et al, 1998), and that the expression of FAAH was low in the cells used by Piomelli et al (1999).…”

Section: Discussionmentioning

confidence: 99%

“…A more exciting possibility, however, is the presence of heterogeneity in AEA uptake mechanisms (for discussion, see Hillard & Jarrahian, 2000). In this respect, Maccarrone et al (1998;2000a,b) found that the cellular uptake rate of AEA in various cells of human origin was greatly enhanced by nitric oxide donors, such as sodium nitroprusside (SNP). In contrast, AEA (and PEA) uptake in Neuro-2a cells and RBL-2H3 cells was not altered by exposure to 5 mM SNP (S.O.P.…”

Section: Discussionmentioning

confidence: 99%

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Characterization of palmitoylethanolamide transport in mouse Neuro‐2a neuroblastoma and rat RBL‐2H3 basophilic leukaemia cells: comparison with anandamide

Jacobsson

Fowler

2001

British J Pharmacology

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1 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. 2 Saturable uptake of PEA and AEA into both cell lines were demonstrated with apparent K M values of 28 mM (PEA) and 10 mM (AEA) in Neuro-2a cells, and 30 mM (PEA) and 9.3 mM (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¯uoride. 3 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 mM, did not a ect AEA uptake in either cell line. AEA, 2-AG, arachidonic acid, R1-methanandamide, D 9 -THC, and cannabidiol inhibited PEA transport in both cell lines. The non-steroidal anti-in¯ammatory drug indomethacin inhibited the AEA uptake but had very weak e ects on the uptake of PEA. 4 From these data, it can be concluded that PEA is transported in to cells both by passive di usion and by a facilitated transport that is pharmacologically distinguishable from AEA uptake.

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Section: Discussionsupporting

confidence: 89%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Characterization of palmitoylethanolamide transport in mouse Neuro‐2a neuroblastoma and rat RBL‐2H3 basophilic leukaemia cells: comparison with anandamide

Jacobsson

Fowler

2001

British J Pharmacology

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“…Electrophoretic analysis of native LOX-1 and its trypsin-digested fragments (20 µg of protein/lane) was performed under reducing conditions on 12% SDS-polyacrylamide gels, using a Mini-Protean II apparatus (Bio-Rad, Richmond, CA) with 0.75 mm spacer arms (25). The prestained molecular mass markers myosin (204 kDa),…”

Section: Methodsmentioning

confidence: 99%

Tryptic Digestion of Soybean Lipoxygenase-1 Generates a 60 kDa Fragment with Improved Activity and Membrane Binding Ability

et al. 2001

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Lipoxygenases are key enzymes in the metabolism of unsaturated fatty acids. Soybean lipoxygenase-1 (LOX-1), a paradigm for lipoxygenases isolated from different sources, is composed of two domains: a ∼30 kDa N-terminal domain and a ∼60 kDa C-terminal domain. We used limited proteolysis and gel-filtration chromatography to generate and isolate a ∼60 kDa fragment of LOX-1 ("mini-LOX"), produced by trypsin cleavage between lysine 277 and serine 278. Mini-LOX was subjected to N-terminal sequencing and to electrophoretic, chromatographic, and spectroscopic analysis. Mini-LOX was found to be more acidic and more hydrophobic than LOX-1, and with a higher content of R-helix. Kinetic analysis showed that mini-LOX dioxygenates linoleic acid with a catalytic efficiency approximately 3-fold higher than that of LOX-1 (33.3 × 10 6 and 10.9 × 10 6 M -1 ‚s -1 , respectively), the activation energy of the reaction being 4.5 ( 0.5 and 8.3 ( 0.9 kJ‚mol -1 for mini-LOX and LOX-1, respectively. Substrate preference, tested with linoleic, R-linolenic, and arachidonic acids, and with linoleate methyl ester, was the same for LOX-1 and mini-LOX, and also identical was the regio-and stereospecificity of the products generated thereof, analyzed by reversed-phase and chiral high-performance liquid chromatography, and by gas chromatography/mass spectrometry. Mini-LOX was able to bind artificial vesicles with higher affinity than LOX-1, but the binding was less affected by calcium ions than was that of LOX-1. Taken together, these results suggest that the N-terminal domain of soybean lipoxygenase-1 might be a built-in inihibitor of catalytic activity and membrane binding ability of the enzyme, with a possible role in physio-(patho)logical conditions.Lipoxygenases are a family of monomeric non-heme, nonsulfur iron dioxygenases which catalyze the conversion of unsaturated fatty acids into conjugated hydroperoxides. Mammalian lipoxygenases have been implicated in the pathogenesis of several inflammatory conditions such as arthritis, psoriasis, and bronchial asthma (1). They have been also implicated in atherosclerosis (2), brain aging (3), HIV infection (4), and kidney disease (5, 6). In plants, lipoxygenases favor germination and participate in the synthesis of traumatin and jasmonic acid and in the response to abiotic stress (7). Recently, they have been also shown to initiate the programmed death (apoptosis) of plant cells (8). Soybean lipoxygenase-1 (LOX-1) 1 is widely used as a prototype for studying the homologous family of lipoxygenases from tissues of different species, both in structural (9-13) and in kinetic (14-18) investigations. The amino acid sequence (19) and three-dimensional structure (9, 10) of LOX-1 have been determined. The overall 839 amino acid residues of LOX-1, with a molecular mass of 93 840 Da, show 2 domains. The first 146 N-terminal amino acid residues form an 8-stranded -barrel, and the remaining 693 residues of the C-terminal domain are organized into 23 helices and 8 -strands. Mammalian lipoxygenases do ...

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“…However, in order to be rapid, this process needs to be driven by controllable and selective mechanisms, such as either a membrane transporter protein, or an intracellular enzymatic process capable of rapidly reducing the intracellular concentration of the two compounds, or both ( Figure 3). Indeed, AEA and 2-AG are taken up by cells via selective, saturable, temperature-dependent and Na þ -independent 'facilitated transport' mechanisms, known as the anandamide membrane transporter(s) (AMT) (Di Marzo et al, 1994;Beltramo et al, 1997;Bisogno et al, 1997a;Hillard et al, 1997;Maccarrone et al, 1998;2000a, b; see also Fowler & Jacobsson et al, 2002 for a review). If one or more proteins is (are) responsible for these mechanisms, however, it (they) has (have) not yet been isolated nor cloned.…”

Section: Cellular Reuptakementioning

confidence: 99%

The endocannabinoid system: a general view and latest additions

Petrocellis

Cascio

Marzo

2004

British J Pharmacology

429

356

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After the discovery, in the early 1990s, of specific G-protein-coupled receptors for marijuana's psychoactive principle D 9 -tetrahydrocannabinol, the cannabinoid receptors, and of their endogenous agonists, the endocannabinoids, a decade of investigations has greatly enlarged our understanding of this altogether new signalling system. Yet, while the finding of the endocannabinoids resulted in a new effort to reveal the mechanisms regulating their levels in the brain and peripheral organs under physiological and pathological conditions, more endogenous substances with a similar action, and more molecular targets for the previously discovered endogenous ligands, anandamide and 2-arachidonoylglycerol, or for some of their metabolites, were being proposed. As the scenario becomes subsequently more complicated, and the experimental tasks to be accomplished correspondingly more numerous, we briefly review in this article the latest 'additions' to the endocannabinoid system together with earlier breakthroughs that have contributed to our present knowledge of the biochemistry and pharmacology of the endocannabinoids.

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Anandamide Hydrolysis by Human Cells in Culture and Brain

Cited by 182 publications

References 44 publications

Characterization of palmitoylethanolamide transport in mouse Neuro‐2a neuroblastoma and rat RBL‐2H3 basophilic leukaemia cells: comparison with anandamide

Characterization of palmitoylethanolamide transport in mouse Neuro‐2a neuroblastoma and rat RBL‐2H3 basophilic leukaemia cells: comparison with anandamide

Tryptic Digestion of Soybean Lipoxygenase-1 Generates a 60 kDa Fragment with Improved Activity and Membrane Binding Ability

The endocannabinoid system: a general view and latest additions

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