Measurement of Icosanoid Precursor Uptake and Release by Intact Cells

This chapter describes the assay for arachidonyl–coenzyme A (CoA) synthetase, assays for the uptake of fatty acid by platelets and cultured cells, an isotopic assay for arachidonate release, and a method for determining the conversion of linoleate to arachidonate by intact cells. Arachidonate uptake can be assayed as radioactivity appearing in platelet phospholipids after incubation with radiolabeled arachidonate. In experiments involving the incubation of platelets with arachidonate for 5 min or more, over 90% of arachidonate in extracted platelet pellets is in phospholipids. Thus, uptake into platelets can be determined without the repeated washing of the cells to remove trapped free arachidonate. The chapter discusses adherent tissue cultured cell. Cells are added to culture dishes and allowed to become adherent to the surface of the dish. At some point after the cells become adherent, the medium is removed and the cells rinsed twice with incubation buffer at 37°. The chapter also illustrates agonist-induced arachidonate release by adherent tissue–cultured cells.