PITFALLS AND SOLUTIONS IN ASSAYING ANANDAMIDE TRANSPORT IN CELLS

The nonspecific binding of anandamide to plastic exhibits many features that could be mistaken as biological processes, thereby representing an important source of conflicting literature data on the uptake and release of this lipophilic substance. In the present study, we sought to minimize the errors associated with the nonspecific binding of AEA to plastic ware, by trying a methodological alternative to traditional protocols. To this aim, we carried out the experiments on glass support (coverslips) instead of plastic, exploiting the limited tendency of hydrophobic molecules to bind the hydrophilic surface of borosilicate glass. In this investigation we used radioactive and immmunocytochemical assays to measure the uptake and the export of AEA. Concerning radioactive assays, in order to compare the results obtained using different supports, we used [3H]AEA in the presence or in the absence of coverslips,. The background, that is the uptake and the export carried out at 4°C, was subtracted from the results obtained at 37°C. As for immunocytochemical assays, cells were treated with bAEA in serum-free medium at 37°C and 4°C, the latter for background subtraction, and the fluorescence was detected by incubating the coverslips with the streptavidin Alexa Fluor 488-conjugated antibody. Although the results obtained using plastic do not differ significantly from those obtained using glass, the new procedure has the advantage to be faster, simpler and more accurate. In fact, the lack of aspecific adsorption of anandamide to the glass surface yields a lower background, a higher precision and accuracy in determination of transport kinetics, especially for the export process. Remarkably, when measured with this procedure the Km constant for anandamide uptake is ∼40% smaller than that found with the traditional method, while the Vmax value does not change significantly. This study describes a fast, simple, and reliable glass-based assay for measuring in vitro AEA uptake and release. This approach is aimed at avoiding some of the artefacts generated by the nonspecific binding of AEA to plastic and the complicated kinetics of AEA/BSA interaction. Considering the fact that AEA does not bind appreciably borosilicate glass, the use of coverslips seems particularly indicated to perform export experiments. More importantly, the new procedure can be also extended to subcellular level, employing b-AEA as a non-radioactive probe to look at anandamide trafficking within intact cells by means of light, electron and fluorescence microscopy techniques.[...]