Direct determination of unbound lipophilic ligands in aqueous solutions

Bojesen, Inge N.

doi:10.1251/bpo93

Cited by 2 publications

(2 citation statements)

References 26 publications

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“…In order to study uptake in the absence of FAAH activity, cells were preincubated for 15 min with 100 nM CAY10400 prior to the addition of AEA. Unbound AEA concentrations were calculated based on the formula (20,31,37 3 H]HT and non-labeled 5-HT at a concentration range of 31.5-1000 nM. Cells were then washed three times with 750 l of ice-cold buffer, 2 ml of liquid scintillation mixture was added, and plates were placed on a shaker overnight.…”

Section: Methodsmentioning

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

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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: Methodsmentioning

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

Self Cite

113

View full text Add to dashboard Cite

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“…If the methyl group of a long‐chain acylcarnitine in the intracellular environment overlaps with the methyl resonance of a triglyceride, after correction for the number of methyl groups per molecule, the concentration of triglycerides would be overestimated by about 2%. The concentration of nonesterified long chain fatty acids in the aqueous phase is negligible (8, 9). Intracellular transfer of long chain fatty acids is mediated by fatty acid binding proteins.…”

mentioning

confidence: 99%