HEPOXILIN-EVOKED INTRACELLULAR REORGANIZATION OF CALCIUM IN HUMAN NEUTROPHILS - A CONFOCAL MICROSCOPY STUDY

Hepoxilin A(3) has previously been shown to cause a rapid dose-depende nt rise in intracellular calcium in intact human neutrophils in suspen sion. Two components have been observed, an initial rapid phase of int racellular calcium rise, followed by a slow decline to plateau levels that remain above the original baseline calcium levels. These changes have been suggested to involve the release of calcium from intracellul ar stores in the ER (initial rapid phase), while the slower rate of de cline (plateau phase) was presumed to be due to calcium influx as it w as abolished in zero calcium extracellular medium. The present study u sed confocal microscopy to examine the response to hepoxilin A(3) at t he subcellular level. Our results show that calcium dynamics in respon se to hepoxilin A(3) varies in different subcellular compartments with in the cell and that hepoxilin A(3) evoked a persistent accumulation o f calcium in organelles. The hepoxilin-evoked calcium sequestration wa s eliminated by prior exposure to CCCP, a mitochondrial uncoupler, CCC P also eliminated the plateau phase of the calcium response in cell su spension, suggesting that this phase was due to mitochondrial accumula tion of calcium rather than calcium influx. Experiments with DiI-loade d cells, a membrane marker, showed that the nuclear calcium was not el evated by hepoxilin addition to the cells. These results demonstrate t hat hepoxilins evoke the release of calcium from the ER which is taken up by the mitochondria where it is tightly sequestered. These results offer an explanation of observations previously made with cell suspen sions in which hepoxilin A(3) was shown to inhibit the calcium mobiliz ing effects of chemotactic agents. (C) 1997 Academic Press.