Biosynthesis of paf-acether factor-acether by human skin fibroblasts in vitro

The synthesis and release of paf-acether by fibroblasts from normal human skin was investigated in vitro. When fibroblasts in suspension (1 X 10(6) cells) were stimulated with 2 microM Ca1+ ionophore A23187 (Io), they synthesized a material that aggregated aspirin-treated washed rabbit platelets and was identified as paf because 1) the platelet aggregation it induced was inhibited by BN 52021, an antagonist of paf putative receptors; 2) the factor was inactivated by phospholipase A2 but was insensitive to lipase from Rhizopus arrhizus; 3) it exhibited the same retention time as synthetic paf during standard and reverse phase HPLC elution. Paf production by fibroblasts occurred as soon as the first min of Io stimulation (287 +/- 92 pg/1 X 10(6) cells), reached a maximum at 5 min (369 +/- 85 pg/1 X 10(6) cells) and decreased thereafter. Half of the fibroblast-produced paf was recovered in supernatants. Addition of exogenous 1-O-alkyl-sn-glycero-3-phosphocholine (lyso-paf) at 0.1 microM and/or acetyl-coenzyme A at 0.1 mM to fibroblasts during Io stimulation enhanced paf production by two- and three-fold, respectively. The paf precursors, i.e., 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine (1-alkyl-2-acyl-GPC) and lyso-paf, were detected in fibroblasts either stimulated with Io or not. These precursors exhibited 80% hexadecyl and 20% octadecyl chains at the sn-1 position of the molecules, as determined by reverse phase HPLC and gas chromatography analysis. The present results are the first to demonstrate the synthesis and release of paf by fibroblasts from normal human skin. Such production within the dermis might account for the development of cutaneous inflammation and for the pathogenesis of many skin disorders.