Platelet-activating factor-induced phosphoinositide metabolism in differentiated U-937 cells in culture

Human monocytic leukemic U-937 cells, when differentiated with dimethylsulfoxide to macrophage-like state, express receptors for platelet-activating factor (PAF). In the differentiated U-937 cells, PAF induced hydrolysis of phosphoinositides and synthesis of inositol phosphates. PAF-induced production of inositol phosphates was rapid, concentration-dependent and was inhibited by a receptor antagonist CV3988, indicating that it was mediated via a specific receptor. In fura-2-loaded, differentiated U-937 cells, PAF induced immediate and concentration-dependent calcium mobilization ( Ca++]i) that was inhibited by CV3988, but not by calcium channel blockers. Addition of an increasing concentration of calcium chelator, ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid, to the medium inhibited a large fraction (approximately 75%) of PAF receptor-induced Ca++]i mobilization thus suggesting the majority of Ca++]i mobilization was originated from extracellular milieu and a small portion (approximately 25%) was originated from intracellular sources. The inositol phosphate production induced by PAF, however, was independent from the extracellular calcium and was not inhibited by the addition of ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid. Neither Ca++]i mobilization or phosphoinositide metabolism in U-937 cells was sensitive to treatment of pertussis toxin, but both types of effects were sensitive to treatment by an inhibitor of phospholipase C, manoalide. These results suggest that in differentiated U-937 cells PAF receptor is coupled through a pertussis toxin-insensitive guanine nucleotide binding protein to a phosphoinositide specific phospholipase C. Inositol-trisphosphate, and possibly diacylglycerol, could be the intracellular messengers for PAF receptor in U-937 cells.