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Journal of Lipid Research, Vol. 47, 561-570, March 2006
Copyright © 2006 by American Society for Biochemistry and Molecular Biology

Effect of an unstirred layer on the membrane permeability of anandamide

Inge N. Bojesen^1,* and Harald S. Hansen

^* Department of Medical Biochemistry and Genetics, Laboratory B, University of Copenhagen, Panum Institute, DK-2200 Copenhagen N, Denmark
Department of Pharmacology and Pharmacotherapy, Danish University of Pharmaceutical Sciences, DK-2100 Copenhagen Ø, Denmark

Published, JLR Papers in Press, December 19, 2005.

¹ To whom correspondence should be addressed. e-mail: norby{at}imbg.ku.dk

To study the effect of an unstirred layer (UL), we have investigated the exchange efflux kinetics of anandamide at 0°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 (k₁) if we know the membrane binding of anandamide, the equilibrium dissociation constant of BSA-anandamide complexes, and the diffusion constant of anandamide. We estimated k₁ to be 3.33 ± 0.27 s^–1. The net flux of [³H]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.

Supplementary key words red blood cell membranes • erythrocyte ghosts • membrane binding • exchange efflux • rate constant of anandamide dissociation from albumin • diffusion coefficient of anandamide

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