Uptake and metabolism of [<sup>3</sup>H]anandamide by rabbit platelets

Fasia, Lambrini; Karava, Vivi; Siafaka-Kapadai, Athanassia

doi:10.1046/j.1432-1033.2003.03724.x

Cited by 34 publications

(33 citation statements)

References 53 publications

(112 reference statements)

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“…51), and only recently extended to include AEA (18,19,22). In our hands, at 37°C the rate of AEA accumulation in RBL-2H3 cells displayed linear uptake kinetics at 25 s, when plotted as a function of total [AEA] in the presence of BSA (Fig.…”

Section: Discussionsupporting

confidence: 51%

“…14 -16). However, in platelets, neuroblastoma, astrocytoma, primary cortical neurons, and erythrocyte ghost membranes it has been suggested that uptake occurs by passive diffusion (13,(17)(18)(19)(20). Experimental variables such as incubation times, inclusion of bovine serum albumin (BSA) in the incubation medium and cell type differences may account for the disparities in results between laboratories (19, 21, 22).…”

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confidence: 99%

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Anandamide Uptake Is Consistent with Rate-limited Diffusion and Is Regulated by the Degree of Its Hydrolysis by Fatty Acid Amide Hydrolase

Kaczocha

Hermann

Glaser

et al. 2006

Journal of Biological Chemistry

113

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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°C. Interestingly, saturation was observed when uptake was plotted using unbound AEA at 37°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°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. Arachidonoyl ethanolamide (AEA)2 is a neuromodulatory lipid that belongs to a family of molecules collectively termed the endocannabinoids. Like ⌬ 9 -tetrahydracannabinol, many of the actions of AEA are mediated through the G-protein-coupled cannabinoid (CB 1 and CB 2 ) and vanilloid (TRPV1) receptors (2-7). AEA signaling is terminated by a rapid reuptake mechanism followed by its hydrolysis into arachidonic acid and ethanolamine primarily by the intracellular enzyme FAAH (8 -10). By metabolizing AEA, FAAH maintains an inward gradient that drives the continued cellular accumulation of AEA (11-13).There is some controversy regarding the processes mediating AEA uptake. It was widely accepted that AEA transport is a selective, saturable process of facilitated diffusion for which there are selective transport inhibitors (For review, see Refs. 14 -16). However, in platelets, neuroblastoma, astrocytoma, primary cortical neurons, and erythrocyte ghost membranes it has been suggested that uptake occurs by passive diffusion (13,(17)(18)(19)(20). Experimental variables such as incubation times, inclusion ...

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Section: Discussionsupporting

confidence: 51%

mentioning

confidence: 99%

Anandamide Uptake Is Consistent with Rate-limited Diffusion and Is Regulated by the Degree of Its Hydrolysis by Fatty Acid Amide Hydrolase

Kaczocha

Hermann

Glaser

et al. 2006

Journal of Biological Chemistry

113

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“…In contrast, AEA is an uncharged lipid capable of traversing the bilayer unaided (10). Accordingly, several studies concluded that AEA uptake occurs by passive diffusion (10)(11)(12)(13)(14)(15), although facilitated diffusion and/or endocytosis have also been proposed (16)(17)(18)(19)(20).…”

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confidence: 99%

Identification of intracellular carriers for the endocannabinoid anandamide

Kaczocha¹,

Glaser

Deutsch³

2009

Proc. Natl. Acad. Sci. U.S.A.

304

332

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The endocannabinoid anandamide (arachidonoyl ethanolamide, AEA) is an uncharged neuromodulatory lipid that, similar to many neurotransmitters, is inactivated through its cellular uptake and subsequent catabolism. AEA is hydrolyzed by fatty acid amide hydrolase (FAAH), an enzyme localized on the endoplasmic reticulum. In contrast to most neuromodulators, the hydrophilic cytosol poses a diffusional barrier for the efficient delivery of AEA to its site of catabolism. Therefore, AEA likely traverses the cytosol with the assistance of an intracellular carrier that increases its solubility and rate of diffusion. To study this process, AEA uptake and hydrolysis were examined in COS-7 cells expressing FAAH restricted to the endoplasmic reticulum, mitochondria, or the Golgi apparatus. AEA hydrolysis was detectable at the earliest measurable time point (

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“…In general, fi ve nonmutually exclusive models have been proposed: i ) passive diffusion ( 23,24 ); ii ) facilitated transport ( 25-27 ); iii ) intracellular traffi cking and sequestration ( 24, 27, 28 ); iv ) endocytosis ( 29, 30 ); and v ) FAAH-mediated uptake ( 31-33 ). These mechanisms might also operAbstract Nonspecifi c binding of anandamide to plastic exhibits many features that could be mistaken as biological processes, thereby representing an important source of confl icting data on the uptake and release of this lipophilic substance.…”

mentioning

confidence: 99%

Pitfalls and solutions in assaying anandamide transport in cells

Oddi¹,

Fezza²,

Catanzaro³

et al. 2010

Journal of Lipid Research

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functions, both in the central nervous system and in the periphery, by binding to type-1 and type-2 cannabinoid receptors ( 1 ), and to transient receptor potential vanilloid 1 (TRPV1) ion channels ( 2, 3 ). Additional targets of AEA also include 5-hydroxytryptamine receptors ( 4 ), GPR55 or "CB3R" ( 5, 6 ), and the nuclear peroxisome proliferatoractivated receptors ␣ ( 7, 8 ) and ␥ ( 9-11 ).AEA is thought to act as an autocrine/paracrine mediator by activating these receptors either on the cell surface (cannabinoid receptors) or within the cell (TRPV1 and peroxisome proliferator-activated receptors). Extracellular AEA signaling is terminated through a two-step process consisting of transport inside the cell and subsequent hydrolysis by fatty acid amide hydrolase (FAAH) ( 12 ). The process whereby AEA is transported across the plasma membrane and within the cell is considered a hot topic, because it is a potential target for treatment of several pathologies associated with endocannabinoid system dysfunctions ( 13-21 ).Despite considerable experimental efforts made to clarify the mechanism of AEA transport, there is not yet general consensus on the identity of the elements responsible for this process [see ( 22 ) for a very recent review]. In general, fi ve nonmutually exclusive models have been proposed: i ) passive diffusion ( 23,24 ); ii ) facilitated transport ( 25-27 ); iii ) intracellular traffi cking and sequestration ( 24, 27, 28 ); iv ) endocytosis ( 29, 30 ); and v ) FAAH-mediated uptake ( 31-33 ). These mechanisms might also operAbstract Nonspecifi c binding of anandamide to plastic exhibits many features that could be mistaken as biological processes, thereby representing an important source of confl icting data on the uptake and release of this lipophilic substance. Herein, we propose an improved method to assay anandamide transport, by using glass slides (i.e., coverslips) as physical support to grow cells. Although the results obtained using plastic do not differ signifi cantly from those obtained using glass, the new procedure has the advantage of being faster, simpler, and more accurate. In fact, the lack of aspecifi c adsorption of anandamide to the glass surface yields a lower background and a higher precision and accuracy in determining transport kinetics, especially for the export process. Remarkably, the kinetic parameters of anandamide uptake obtained with the old and the new procedures may be similar or different depending on the cell type, thus demonstrating the complexity of the interference of plastic on the transport process. In addition, the novel procedure is particularly suitable for visualization and measurement of anandamide transport in intact cells by using a biotinylated derivative in confocal fl uorescence microscopy.

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Uptake and metabolism of [³H]anandamide by rabbit platelets

Cited by 34 publications

References 53 publications

Anandamide Uptake Is Consistent with Rate-limited Diffusion and Is Regulated by the Degree of Its Hydrolysis by Fatty Acid Amide Hydrolase

Anandamide Uptake Is Consistent with Rate-limited Diffusion and Is Regulated by the Degree of Its Hydrolysis by Fatty Acid Amide Hydrolase

Identification of intracellular carriers for the endocannabinoid anandamide

Pitfalls and solutions in assaying anandamide transport in cells

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Uptake and metabolism of [3H]anandamide by rabbit platelets

Cited by 34 publications

References 53 publications

Anandamide Uptake Is Consistent with Rate-limited Diffusion and Is Regulated by the Degree of Its Hydrolysis by Fatty Acid Amide Hydrolase

Anandamide Uptake Is Consistent with Rate-limited Diffusion and Is Regulated by the Degree of Its Hydrolysis by Fatty Acid Amide Hydrolase

Identification of intracellular carriers for the endocannabinoid anandamide

Pitfalls and solutions in assaying anandamide transport in cells

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Uptake and metabolism of [³H]anandamide by rabbit platelets