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-hydroperoxyeicosa (5Z,8Z,10E,14Z) tetraenoic acid), on the release of intracellular calcium in intact human neutrophils using the INDO-1 AM fluorescent dye technique. Both products dose dependently stimulate intracellular release, with 12-HETE being more powerful than 12-HPETE. 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 slightly more active than the (12R) isomer. The laser potency of 12-HPETE may be due to its conversion into the less active hepoxilins as incubation of neutrophils with (12S/R)-HPETE in a nonradioactive assay, using fluorescent ADAM esters of the products, generated mostly hepoxilin A3 (8-hydroxy-(11S,12S) epoxyeicosa (5Z,9E,14Z)trienoic acid), indicative of an enzymatic process. In contrast, boiled neutrophil preparations converted 12-HPETE primarily into hepoxilin B3 which previously showed to be derived nonenzymatically. This data demonstrates that 12-HETE, known to be generated in significant amounts by platelets, can act transcellularly to modify intracellular concentrations of calcium in neutrophils. This may in turn affect the responsiveness of these cells to other chemotactic factors.