Abstract
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.
Highlights
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)
Anandamide is found in mammalian tissues, where it accumulates upon tissue injury [1,2,3]
When studying the transport of lipophilic compounds whose membrane translocation is rapid, it is important to consider the effect of an unstirred layer (UL) around cells, because it may be a rate-limiting barrier for transport [23, 24]
Summary
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). 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 evidence for the existence of an anandamide transporter in the plasma membrane comes from saturable uptake kinetics in cells and from the use of inhibitors, but to date no carrier proteins have been isolated. Studies have reported both saturable and nonsaturable uptake kinetics using total anandamide concentration as a variable [17, 20,21,22]. Finding saturable kinetics may not be a definitive proof for a carrier-mediated process
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