Revised Manuscript Received July 8, 1991 abstract: Our studies assessed the effects of increases in intracellular calcium concentrations ([Ca2+]¡) on leukotriene synthesis and membrane translocation of 5-lipoxygenase (5LO). The calcium ionophore ionomycin and the tumor promoter thapsigargin stimulated leukotriene production and translocation of 5-lipoxygenase to the membrane. Both agents elicited prolonged rises in [Ca2+]¡. Leukotriene C4 production associated with [Ca2+]¡ in cells stimulated with various concentrations of ionomycin and thapsigargin suggests that a threshold [Ca2+]¡ level of approximately 300-400 nM is required. In the absence of extracellular Ca2+, both the ionomycin-and thapsigargin-induced rises in [Ca2+]¡ were transient, indicating that the prolonged [Ca2+]¡ elevation is dueto an influx of extracellular Ca2+. Addition of EGTA to the external medium before, or at different times during, the treatment with ionomycin or thapsigargin instantaneously inhibited 5LO translocation and leukotriene synthesis, indicating that Ca2+ influx plays an essential role in 5LO membrane translocation and leukotriene synthesis. No leukotriene production was detected when cells were stimulated by a physiological stimulus of leukotriene D4. The addition of 100 nM leukotriene D4 triggered peakrises in [Ca2+]¡ that were comparable to those achieved by the ionomycin and thapsigargin. However, the leukotriene D4 induced rise was transient and rapidly declined to a lower but still elevated steady-state level, which was attributed to Ca2+ influx. Stimulation with 100 nM leukotriene D4 for 15 s increased the cellular levels of 1, 4, 5-inositol triphosphate (IP3), 1, 3, 4-IP3, and 1, 3, 4, 5-inositol tetraphosphate (IP4). In contrast, 100 nM thapsigargin had no effect on generating inositol phosphate after 15, 60, or 300 s of treatment. These results argue against an essential role for inositol phosphates in leukotriene synthesis and indicatethat the stimulation of 5-lipoxygenase membrane translocation and leukotriene synthesis is a consequence of a sustained increase in [Ca2+]¡ resultingfrom an influx of external Ca2+.

¡ Studies with a variety of cellsystems have led to the view that a rise in [Ca2+]¡'is an obligatory step in thesynthesis of leukotrienes. The main evidence for thisconclusion is sum-marized as follows: First, leukotriene (LT) synthesis can be obtained after binding of agonists to cell surface receptors, which results in an increase of [Ca2+]¡. For example, ma-crophages synthesize LTC4in response to several Ca2+-mo-bilizing stimuli such as opsonized zymosan (Humes et al., 1982; Tripp et al., 1985) and immunoglobulin E complexed with antigen (Rouzeret al., 1982). Second, calcium ionophore A23187 induces leukotriene synthesis (Borgeat et al., 1979), presumably through a rise in [Ca2+]¡. Third, at least two enzymes in the leukotriene synthesis pathway require Ca2+: phospholipase A2, for release of arachidonic acid (Wijkander & Sundler, 1989; Leslie et al., 1988), and 5-lipoxygenase (5LO), for synthesis of 5-hydroperoxyeicosatetraenoic acid and LTA4 (Jakschik & Lee, 1980). In addition, Ca2+ regulates