Intracellular Ca2+, inositol 1,4,5-trisphosphate and additional signalling in the stimulation by platelet-activating factor of prostaglandin E2 formation in P388D1 macrophage-like cells.
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 the 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 intracellular 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 physiologically relevant response of the cell population. Priming the cells with LPS was required for PAF to stimulate PGE2 formation, yet LPS affected 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 Ca2+ transient, the release of Ins(1,4,5)P3 and the formation of PGE2 induced by PAF were inhibited in cells pretreated with pertussis toxin. Buffering the [Ca2+]i with intracellular BAPTA [bis-(o-aminophenoxy)ethane-NNN'N'-tetra-acetic acid] blocked the PAF-stimulated rise in [Ca2+]i and PGE2 formation. Removal of extracellular Ca2+ during PAF stimulation prevented the influx of Ca2+, but did not affect the initial [Ca2+]i transient, nor did it inhibit PGE2 formation. Under the same conditions, ionomycin stimulated an identical [Ca2+]i transient, but, in contrast with PAF stimulation, no PGE2 formation was observed. PGE2 production could be rescued by prompt subsequent addition of PAF, which caused no further [Ca2+]i change on its own. These results show that the transient initial rise in [Ca2+]i, produced either by PAF via the formation of Ins(1,4,5)P3 or directly by ionomycin, is necessary, but not sufficient for the formation of PGE2 in LPS-primed P388D1 cells. Furthermore, we have demonstrated for the first time that PAF triggers a second signal that is not mediated by a change in [Ca2+]i. However, both signals are required to induce PGE2 formation.
Asmis
R
R
Department of Chemistry (0601), University of California, San Diego, La Jolla 92093.
Randriamampita
C
C
Tsien
R Y
RY
Dennis
E A
EA
D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D015232:Dinoprostone / Q000096:biosynthesis*; D004533:Egtazic Acid / Q000494:pharmacology; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D015759:Ionomycin / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000378:metabolism*; D037342:Pertussis Toxin; D010972:Platelet Activating Factor / Q000494:pharmacology*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D015398:Signal Transduction; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D008070:Lipopolysaccharides; D010972:Platelet Activating Factor; D010566:Virulence Factors, Bordetella; D004533:Egtazic Acid; D015759:Ionomycin; D015544:Inositol 1,4,5-Trisphosphate; D037342:Pertussis Toxin; D011505:Protein-Tyrosine Kinases; D015232:Dinoprostone; D002118:Calcium