TRANSPORT OF ARACHIDONIC-ACID ACROSS THE NEUTROPHIL PLASMA-MEMBRANE VIA A PROTEIN-FACILITATED MECHANISM

Arachidonic acid is the rate-limiting substrate in the biosynthesis of leukotrienes in activated neutrophils. Liberation of arachidonate fro m 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 usin g intact polymorphonuclear neutrophils and inside-out plasma membrane vesicles thereof. Here we show that the cellular uptake of arachidonat e is energy dependent with an energy of activation (E-A) 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, ch emical protein modifying reagents, anion transport inhibitors, and, mo st notably, toward several structurally unrelated leukotriene B-4 rece ptor antagonists with IC50 values in the range of 16-44 mu M. The inhi bitors did not inhibit the diffusional uptake of methyl arachidonate i nto neutrophils and inside-out plasma membrane vesicles, indicating th at a transport protein is required for the rapid uptake of the free ac id but not for the uptake of the ester. Other long-chain fatty acids d id compete with the uptake of arachidonate in both assay systems, wher eas leukotriene Bq did not. This study documents a novel protein-facil itated transport mechanism for arachidonate in neutrophils, potentiall y involved in transcellular eicosanoid biosynthesis and sPLA(2)-mediat ed arachidonate signaling in neutrophils.