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 ...

The endocannabinoid anandamide is of lipid nature and may thus bind to albumin in the vascular system, as do fatty acids. The knowledge of the free water-phase concentration of anandamide is essential for the investigations of its transfer from the binding protein to cellular membranes, because a water-phase shuttle of monomers mediates such transfers. We have used our method based upon the use of albumin-filled red cell ghosts as a dispersed biological "reference binder" to measure the water-phase concentrations of anandamide. These concentrations were measured in buffer (pH 7.

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Thermodynamics and kinetics of serum albumin binding of long-chain fatty acids (FA) with more than 14 carbon atoms are important in all chemical studies of FA in water. Furthermore, the energetics of this process should substantiate the molecular model (Brown, J. R.; Shockley, P. Lipid-Protein Interactions; John Wiley & Sons: New York, 1989; Vol. 1, pp 25-68). At pH 7.3, bovine serum albumin (BSA) has three equivalent high affinity binding sites for FA to BSA molar ratios below 2 at temperatures between 273 and 311 K. The corresponding Gibbs free energies (∆G°) of six FA transfers from buffer, ionic strength 0.173 M, to BSA are identical with those of transfers to heptane of FA excepting the head groups -CH 2 -COO -(Smith, R.; Tanford, C. Proc. Natl. Acad. Sci. U.S.A. 1973, 70, 289-293), suggesting similar binding free energy of the anionic head group in buffer and in BSA, corroborating NMR studies (Cistola, D. P.; Small, D. M.; Hamilton, J. A. J. Biol. Chem. 1989, 262, 10980-10985). From ∆H°and ∆G°of FA transfers from water to BSA and to aliphatic hydrocarbons, we calculate T∆S°and ∆H°of the FA hydrocarbon chains transfers from aliphatic hydrocarbons to BSA, with complete entropy/enthalpy compensation (∆G°≈ 0). The values vary between FA but corroborate known conformational changes of BSA when binding FA. The kinetics of palmitate and oleate bindings reveal transition states of FA with low entropy as in water and higher enthalpies corresponding to carboxyl-group bindings by hydrogen bonds in water. Thus, FA are bound in BSA by combined electrostatic anionic head group bindings and van der Waals bindings of the hydrocarbon tails. The ∆G°'s of transfer from buffer are entirely accounted for by transfer of the hydrocarbon tail, making predictable the equilibrium constants of FA-BSA complexes in buffer at pH 7.3.