OUTER-MEMBRANE PORINS FROM GRAM-NEGATIVE BACTERIA STIMULATE PLATELET-ACTIVATING-FACTOR BIOSYNTHESIS BY CULTURED HUMAN ENDOTHELIAL-CELLS

Porins are a family of hydrophobic proteins located in the outer membr ane of the cell wall in Gram-negative bacteria. The effect of porins o n the biosynthesis of platelet-activating factor (PAF) by cultured hum an umbilical-cord-vein-derived endothelial cells (HUVEC) was investiga ted. The results demonstrate that porins were able to induce a dose-de pendent synthesis of PAF in HUVEC. PAF, synthesized after stimulation with porins, was mainly cell associated and the synthesis peaked at 15 min, decreasing rapidly thereafter. Experiments with radiolabeled pre cursors demonstrated that PAF, a -0-alkyl-2-acetyl-sn-glyceryl-3-phosp horylcholine, was synthesized via the remodeling pathway involving the acetylation of 1-O-alkyl-2-lyso-sn-glyceryl-3-phosphorylcholine (2-ly soPAF) generated from 1-0-alkyl-2-acyl-sn-glyceryl-3-phosphorylcholine by phospholipase-A2 activity. The activation of phospholipase A2 in H UVEC stimulated by porins was detected by observing the mobilization o f [C-14]arachidonic acid. In addition, the activity of acetyl-CoA:1-al kyl-sn-glycero-3-phosphorylcholine 2-O-acetyltransferase was transient ly increased in porin-stimulated HUVEC and, after incubation with [H-3 ]CoASAc or [H-3]acetate, the [H-3]acetyl group was incorporated into n ewly synthesized PAF. Porins, by forming transmembrane channels, induc ed a sustained influx of extracellular Ca-45(2+) into the cytosol. The activation of PAF synthesis by porins depended on this influx rather than on intracellular calcium mobilization, since PAF synthesis did no t occur in the absence of extracellular Ca2+.