INTRACELLULAR CA2-TRISPHOSPHATE AND ADDITIONAL SIGNALING IN THE STIMULATION BY PLATELET-ACTIVATING-FACTOR OF PROSTAGLANDIN-E(2) FORMATION IN P388D(1) MACROPHAGE-LIKE CELLS(, INOSITOL 1,4,5)

In the P388D1 macrophage-like cell line, phospholipase A2 activity and prostaglandin production are stimulated by platelet-activating factor (PAF) and bacterial lipopolysaccharide (LPS). We have investigated th e role of Ins(1,4,5)P3 and Ca2+ in signal transduction of PAF-induced prostaglandin E2 (PGE2) formation in these cells. The role of Ca2+ in the activation mechanism was studied by fluorescence imaging of intrac ellular Ca2+ in individual adherent cells and by determining the PGE2 production in the same population of cells. This new approach enabled us to correlate directly events on the single-cell level with a physio logically relevant response of the cell population. Priming the cells with LPS was required for PAF to stimulate PGE2 formation, yet LPS aff ected neither the intracellular free Ca2+ concentration ([Ca2+]i) nor the PAF-induced rise in [Ca2+]i. In addition, basal and PAF-stimulated Ins(1,4,5)P3 levels were not affected by LPS priming. However, the Ca 2+ transient, the release of Ins(1,4,5)P3 and the formation of PGE2 in duced by PAF were inhibited in cells pretreated with pertussis toxin. Buffering the [Ca2+]i with intracellular BAPTA [bis-(o-aminophenoxy)et hane-NNN'N'-tetra-acetic acid] blocked the PAF-stimulated rise in [Ca2 +]i and PGE, formation. Removal of extracellular Ca2+ during PAF stimu lation prevented the influx of Ca2+, but did not affect the initial [C a2+]i transient, nor did it inhibit PGE2 formation. Under the same con ditions, ionomycin stimulated an identical [Ca2+]i transient, but, in contrast with PAF stimulation, no PGE2 formation was observed. PGE2 pr oduction could be rescued by prompt subsequent addition of PAF, which caused no further [Ca2+]i change on its own. These results show that t he transient initial rise in [Ca2+]i, produced either by PAF via the f ormation of Ins(1,4,5)P3 or directly by ionomycin, is necessary, but n ot sufficient for the formation of PGE2 in LPS-primed P388D1 cells. Fu rthermore, we have demonstrated for the first time that PAF triggers a second signal that is not mediated by a change in [Ca2+]i. However, b oth signals are required to induce PGE2 formation.