12-HETE AND 12-HPETE POTENTLY STIMULATE INTRACELLULAR RELEASE OF CALCIUM IN INTACT HUMAN NEUTROPHILS

We describe here the effects of two 12-lipoxygenase products, 12-HETE (12-hydroxyeicosa (5Z,8Z,10E,14Z) tetraenoic acid) and 12-HPETE (12-hy droperoxyeicosa (5Z,8Z,10E,14Z) tetraenoic acid), on the release of in tracellular calcium in intact human neutrophils using the INDO-1 AM fl uorescent dye technique. Both products dose dependently stimulate intr acellular calcium release, with 12-HETE being more powerful than 12-HP ETE. The threshold concentration for 12-HETE was 5 ng/ml (1.5 x 10(-8) M), while that for 12-HPETE was 10 ng/ml. The (12S) regioisomer was s lightly more active than the (12R) isomer. The lesser potency of 12-HP ETE may be due to its conversion into the less active hepoxilins as in cubation of neutrophils with (12S/R)-HPETE in a nonradioactive assay, using fluoresecent ADAM esters of the products, generated mostly hepox ilin A3 (8-hydroxy-(11S,12S) epoxyeicosa (5Z,9E,14Z)trienoic acid), in dicative of an enzymatic process. In contrast, boiled neutrophil prepa rations converted 12-HPETE primarily into hepoxilin B-3 which we previ ously showed to be derived nonenzymatically. This data demonstrates th at 12-HETE, known to be generated in significant amounts by platelets, can act transcellularly to modify intracellular concentrations of cal cium in neutrophils. This may in turn affect the responsiveness of the se cells to other chemotactic factors.