Reversible, temperature-dependent, and AM404-inhibitable adsorption of anandamide to cell culture wells as a confounding factor in release experiments

Relatively little is known about the process whereby the endocannabinoid anandamide (AEA) is released from cells. A simple way of studying this process is to sample the appearance in the medium of tritium following preloading of cells with [3 H]AEA under conditions where its metabolism is prevented. However, this approach may be complicated by the ability of AEA to be adsorbed reversibly to the cell culture wells. In the present study, it is found that cell culture wells adsorb almost half of the added AEA in a manner prevented by fatty acid-free bovine serum albumin, and by the prototypical uptake inhibitors AM404 and VDM11 with IC50 values of 3 and 1 muM, respectively. After incubation followed by washing of the plates, AEA is released into the medium from the wells by a first order process (k similar to 0.1 min(-1)) that is temperature-dependent and increased by AM404 and fatty acid-free bovine serum albumin. When assays were run with 0.15% fatty acid-free bovine serum albumin during the loading, washing and release phases of the assay, the release from the well was greatly reduced and a first order, temperature-sensitive release from C6 glioma cells could be unmasked. It is concluded that the reversible adsorption of AEA by cell culture wells can be a confounding factor in release experiments. (C) 2004 Elsevier B.V. All rights reserved.