The involvement of extracellular calcium in the formation of 5‐lipoxygenase metabolites by human polymorphonuclear leukocytes

We have addressed the question why in the presence of a Ca 2+ ionophore human polymorphonuclear leukocytes generate leukotrienes in high yields, but in only low amounts after stimulation by receptor agonists like fMLF (fM, formylmethionine), leukotriene B 4 or platelet‐activating factor (PAF), although a significant release of intracellular calcium can be measured. Using ionomycin we can show that from the two enzymes involved, phospholipase A 2 and 5‐lipoxygenase, the first requires a threshold level of about 350–400 nM calcium whereas 5‐lipoxygenase shows a linear dependence on calcium and saturates at this concentration. Our data indicate that the Ca 2+ requirement of phospholipase A 2 can only be met by an additional influx of extracellular calcium, whereas 5‐lipoxygenase will operate already at levels provided by intracellular stores. Consequently, the complexing of extracellular calcium by EGTA stops phospholipase A 2 activity immediately, whereas added arachidonate can be still adequately metabolized by intracellular Ca 2+ release triggered by fMLF or PAF. Interestingly, PAF shows a stronger extracellular component in its Ca 2+ transient than fMLF, and also generates more 5‐lipoxygenase metabolites. However, a clear correlation between the amount of 5‐lipoxygenase metabolites and the extracellular Ca 2+ signal was lacking, since maximal activity was achieved before the bulk of the extracellular calcium was monitored. Ca 2+ influx after PAF stimulation could be blocked after 2 min by EGTA, but a further increase in the formation of 5‐lipoxygenase metabolites was observed. In contrast ionomycin‐elicited 5‐lipoxygenase activity could be stopped at any time shortly after EGTA addition.