Direct determination of unbound lipophilic ligands in aqueous solutions.

Due to their hydrophobic nature, lipophilic compounds are always bound to proteins when transported in the organism. The transfer of such compounds between their binding proteins and cells as well as intracellular trafficking is mediated by a very low water-phase concentration of monomers. The use of protein filled resealed red cell membranes (erythrocyte ghosts) as semipermeable bags enables us to determine directly such water-phase concentrations in a biological system where the lipophilic compound is in equilibrium with the compound bound to its binding protein. Equilibrium dissociation constants (K(d)'s) and number of binding sites are determined by regression analyses of data. We describe the method with the hydrophobic anion arachidonate and the neutral N-arachidonoylethanolamide as examples.

Effect of an unstirred layer on the membrane permeability of anandamide.

To study the effect of an unstirred layer (UL), we have investigated the exchange efflux kinetics of anandamide at 0 degrees C, pH 7.3, from albumin-free as well as from albumin-filled human red blood cell ghosts to media of various BSA concentrations ([BSA](o)). The rate constant (k(m)) of unidirectional flux from the outer membrane leaflet to BSA in the medium increased with the square root of [BSA](o) in accordance with the existence of a UL, which is a water layer adjacent to the membrane that is not subject to the same gross mixing that takes place in the rest of the medium. From k(m), it is possible to calculate the rate constant of anandamide dissociation from BSA (k(1)) if we know the membrane binding of anandamide, the equilibrium dissociation constant of BSA-anandamide complexes, and the diffusion constant of anandamide. We estimated k(1) to be 3.33 +/- 0.27 s(-1). The net flux of [(3)H]anandamide is balanced by an equal and opposite movement of nonradioactive anandamide in exchange efflux experiments. This means that our results are also valid for uptake. We show that for anandamide with rapid membrane translocation, UL causes a significant resistance to cellular uptake. Depicting the rate of anandamide uptake as a function of equilibrium water phase concentrations results in a parabolic uptake dependence. Such apparent "saturation kinetics" is often interpreted as indicating the involvement of transport proteins. The validity of such an interpretation is discussed.

Anandamide uptake is consistent with rate-limited diffusion and is regulated by the degree of its hydrolysis by fatty acid amide hydrolase.

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.

Membrane transport of anandamide through resealed human red blood cell membranes.

The use of resealed red blood cell membranes (ghosts) allows the study of the transport of a compound in a nonmetabolizing system with a biological membrane. Transmembrane movements of anandamide (N-arachidonoylethanolamine, arachidonoylethanolamide) have been studied by exchange efflux experiments at 0 degrees C and pH 7.3 with albumin-free and albumin-filled human red blood cell ghosts. The efflux kinetics is biexponential and is analyzed in terms of compartment models. The distribution of anandamide on the membrane inner to outer leaflet pools is determined to be 0.275 +/- 0.023, and the rate constant of unidirectional flux from inside to outside is 0.361 +/- 0.023 s(-1). The rate constant of unidirectional flux from the membrane to BSA in the medium ([BSA]o) increases with the square root of [BSA]o in accordance with the theory of an unstirred layer around ghosts. Anandamide passed through the red blood cell membrane very rapidly, within seconds. At a molar ratio of anandamide to BSA of <1, membrane binding of anandamide increases with increasing temperatures between 0 degrees C and 37 degrees C, and the equilibrium dissociation constants are in the nanomolar range. The nature of membrane binding and the mechanism of membrane translocation are discussed.

Binding of anandamide to bovine serum albumin.

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 7.3) in equilibrium with anandamide bound to BSA inside resealed human red cell membranes at low molar ratios below one. Data were obtained at 0 degrees C, 10 degrees C, 23 degrees C, and 37 degrees C. The equilibrium dissociation constant (Kd) increases with temperature from 6.87 +/- 0.53 nM at 0 degrees C to 54.92 +/- 1.91 nM at 37 degrees C. Regression analyses of the data suggest that BSA has one high-affinity binding site for anandamide at all four temperatures. The free energy of anandamide binding (DeltaG0) is calculated to -43.05 kJ mol-1 with a large enthalpy (DeltaH0) contribution of -42.09 kJ mol-1. Anandamide has vasodilator activity, and the binding to albumin may mediate its transport in aqueous compartments.