HEPOXILIN-A(3) INHIBITS THE RISE IN FREE INTRACELLULAR CALCIUM EVOKEDBY FORMYL-METHIONYL-LEUCYL-PHENYLALANINE, PLATELET-ACTIVATING-FACTOR AND LEUKOTRIENE-B(4)

We have previously shown that the hepoxilins are capable of increasing the intracellular free concentration of calcium ([Ca2+]i) in human ne utrophils through a pertussis toxin-sensitive, extracellular calcium-i ndependent pathway involving the mobilization of calcium from internal stores. A subsequent hepoxilin-induced and extracellular calcium-depe ndent influx of calcium is observed. In an effort to investigate furth er the role of these compounds in the human neutrophil, we investigate d their potential effects on the action of known agonists such as form yl-methionine-leucine-phenylalanine (fMLP). platelet-activating factor (PAF) and leukotriene B4 (LTB4) on the mobilization of calcium. Hepox ilis dose-dependently inhibited the increases in [Ca2+], induced by fM LP, PAF and LTB4. The hepoxilin concentration required for inhibition was around 100 ng/ml (3 x 10(-7) M). This concentration of hepoxilin d id not cause any measurable change in [Ca2+]i. The extent of inhibitio n of the agonist-evoked rise in [Ca2+]i by hepoxilins was proportional to the increase in the calcium response evoked by hepoxilin beyond it s threshold concentration. Additional experiments were carried out to investigate the mechanism for the hepoxilin effect. Using calcium-free medium and in the presence of sufficient amounts of thapsigargin (200 ng/ml) to maximally block the calcium pump (thereby achieving a const ant rate of calcium leakage from stores), hepoxilin A3 increased furth er this rate of calcium from stores. Its potential (additional) thapsi gargin-like action in blocking the pump, however, cannot be ruled out by these experiments. These observations suggest that the hepoxilins m ay serve an important negative regulatory function in the agonist-indu ced mobilization of calcium in these cells by depleting calcium stores .