Effect of unstirred layer on membrane permeability of anandamide

Inge N. Bojesen and Harald S. Hansen

of Medical Biochemistry and Genetics, Lab. B, University of Copenhagen, Copenhagen DK-2200 N

Corresponding Author: norby{at}imbg.ku.dk

In order 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 ghost to media of various bovine serum albumin concentrations [BSA]o.The rate constant (km) of unidirectional flux from the outer membrane leaflet to BSA in the medium increases with square root of [BSA]o in accordance with existence of an UL, which is a water-layer adjacent to the mambrane that is not subject to the same gross mixing that take place in the rest of the medium. From km it was possible to calculate the rate constant of anandamide dissociation from BSA (k1) on condition that we know membrane binding of anandamide , the equilibrium dissociation constant (Kd) of BSA-anandamide complexes and the diffusion constant of anandamide.We estimate k1 to 3.33-+0.27 s-1. The net flux of 3H-anandamide is balanced by an equal and opposite movement of non-radioactive anandamide in exchange efflux experiments. It means that results are valid also for uptake.We show that for anandamide with rapid membrane translocation,UL causes a significant resistance to the cellular uptake. Depicting rate of anandamide uptake as a function of equilibrium water-phase concentrations gives a parabolic uptake dependency.Such apparent"saturation kinetic"is often interpreted as an involvement of transport proteins. The validity of such an interpretation is discussed.

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