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 membrane of the cell wall in Gram-negative bacteria. The effect of porins on the biosynthesis of platelet-activating factor (PAF) by cultured human umbilical-cord-vein-derived endothelial cells (HUVEC) was investigated. The results demonstrate that porins were able to induce a dose-dependent 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 precursors demonstrated that PAF, a 1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine, was synthesized via the remodeling pathway involving the acetylation of 1-O-alkyl-2-lyso-sn-glyceryl-3-phosphorylcholine (2-lysoPAF) generated from 1-O-alkyl-2-acyl-sn-glyceryl-3-phosphorylcholine by phospholipase-A2 activity. The activation of phospholipase A2 in HUVEC stimulated by porins was detected by observing the mobilization of [14C]arachidonic acid. In addition, the activity of acetyl-CoA:1-alkyl-sn-glycero-3-phosphorylcholine 2-O-acetyltransferase was transiently increased in porin-stimulated HUVEC and, after incubation with [3H]CoASAc or [3H]acetate, the [3H]acetyl group was incorporated into newly synthesized PAF. Porins, by forming transmembrane channels, induced a sustained influx of extracellular 45Ca2+ into the cytosol. The activation of PAF synthesis by porins depended on this influx rather than on intracellular calcium mobilization, since PAF synthesis did not occur in the absence of extracellular Ca2+.
Istituto di Micobiologia, Facoltà di Medicina e Chirurgia, Seconda Università di Napoli, Italy.
M A
MA
Tufano
L
L
Biancone
F
F
Rossano
C
C
Capasso
A
A
Baroni
A
A
De Martino
E L
EL
Iorio
L
L
Silvestro
G
G
Camussi
D000123:Acetyltransferases / Q000378:metabolism; D001425:Bacterial Outer Membrane Proteins / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism*; D006090:Gram-Negative Bacteria / Q000737:chemistry*; D006801:Humans; D010741:Phospholipases A / Q000378:metabolism; D054467:Phospholipases A2; D010972:Platelet Activating Factor / Q000031:analogs & derivatives / Q000096:biosynthesis* / Q000378:metabolism; D018272:Porins; D012486:Salmonella typhimurium; D014471:Umbilical Veins
D001425:Bacterial Outer Membrane Proteins; C029271:O-deacetyl platelet activating factor; D010972:Platelet Activating Factor; D018272:Porins; D000123:Acetyltransferases; C046035:1-alkylglycerophosphocholine acetyltransferase; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium