PLATELET-ACTIVATING-FACTOR BIOSYNTHESIS INDUCED BY VARIOUS STIMULI INHUMAN HACAT KERATINOCYTES

Platelet-activating factor (PAF) is a potent inflammatory mediator tha t is thought to play a role in cutaneous inflammation. These studies u sed mass spectrometry to examine the molecular species of PAF precurso r glycerophosphocholine lipids (GPC) as well as the biosynthesis of PA F and other sn-2 acetyl-GPC in a human keratinocyte-derived cell line (HaCaT keratinocytes). Approximately 28% of HaCaT keratinocyte GPC con sisted of 1-alkyl. species, and the relative amounts of the sn-1 allyl constituents of the PAF precursor 1-alkyl-2-acyl-GPC were as follows: hexadecyl > octadecenyl > octadecyl. Ionophore (A23187)-stimulated Ha CaT keratinocytes synthesized both PAF (1-hexadecyl, 1-octadecenyl, an d 1-octadecyl species) and less potent 1-acyl analogs (1-palmitoyl, 1- oleoyl, and 1-stearoyl species). PAF production was rapid and maximal by 10 min, The major species of sn-2 acetyl-GPC at 2.5 min were 1-hexa decyl-2-acetyl-GPC (2.2 ng/10(6) cells) and 1-palmitoyl-2-acetyl-GPC ( 2.4 ng/10(6) cells), HaCaT keratinocytes also synthesized PAF and 1-ac yl PAF analogs when stimulated with the peptide growth factor endothel in-1 and the nonhydrolyzable PAF receptor agonist carbamyl-PAF. Both 1 -hexadecyl-2-acetyl-GPC and 1-palmitoyl-2-acetyl-GPC stimulated intrac ellular calcium mobilization in HaCaT cells, indicating that these sn- 2 acetyl-GPC act in autocrine fashion, These studies revealed that the human keratinocyte-derived cell line HaCaT can synthesize significant amounts of PAF and 1-acyl analogs in vitro from both nonspecific (A23 187) and specific (endothelin-1, carbamyl-PAF) stimulation, suggesting a role for this inflammatory lipid mediator in keratinocyte pathophys iology.