Transport of Arachidonic Acid across the Neutrophil Plasma Membrane via a Protein-Facilitated Mechanism

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Abstract
Arachidonic acid is the rate-limiting substrate in the biosynthesis of leukotrienes in activated neutrophils. Liberation of arachidonate from intracellular membranes and uptake of exogenous arachidonate are the two principal mechanisms by which the cell can increase the level of this substrate. We investigated arachidonate uptake and export by using intact polymorphonuclear neutrophils and inside-out plasma membrane vesicles thereof. Here we show that the cellular uptake of arachidonate is energy dependent with an energy of activation (EA) of 10.0 kcal/mol and half-saturated at an arachidonate concentration of 4.8 nmol/mg of cell protein. Protein-facilitated transport of arachidonate across the plasma membrane in either direction is sensitive to proteases, chemical protein modifying reagents, anion transport inhibitors, and, most notably, toward several structurally unrelated leukotriene B4 receptor antagonists with IC50 values in the range of 16-44 microM. The inhibitors did not inhibit the diffusional uptake of methyl arachidonate into neutrophils and inside-out plasma membrane vesicles, indicating that a transport protein is required for the rapid uptake of the free acid but not for the uptake of the ester. Other long-chain fatty acids did compete with the uptake of arachidonate in both assay systems, whereas leukotriene B4 did not. This study documents a novel protein-facilitated transport mechanism for arachidonate in neutrophils, potentially involved in transcellular eicosanoid biosynthesis and sPLA2-mediated arachidonate signaling in neutrophils.