Porins and lipopolysaccharide stimulate platelet activating factor synthesis by human mesangial cells.
Porins, a family of hydrophobic proteins located in the outer membrane of the cell wall of gram-negative bacteria and lipopolysaccharide (LPS), were shown to stimulate the synthesis of platelet activating factor (PAF), a phospholipid mediator of inflammation and endotoxic shock, by cultured human glomerular mesangial cells (MC). The synthesis of PAF induced by porins was rapid (peak at 20 min) and independent either from contamination by LPS or from generation of an endotoxin-induced cytokine such as tumor necrosis factor (TNF) since it was not prevented by cycloheximide, an inhibitor of protein synthesis or anti-TNF blocking antibodies. LPS also stimulated PAF synthesis by MC. However, the kinetic of PAF synthesis induced by LPS was biphasic with an early and transient peak at 10 minutes and a second and sustained peak at three to six hours. This second peak required an intact protein synthesis and was prevented by anti-TNF antibodies, suggesting the dependency on LPS-induced synthesis of TNF. Experiments with labeled precursors demonstrated that in MC, either after stimulation with porins or LPS, PAF was synthesized via the remodeling pathway that involves acetylation of 1-0-alkyl-sn-glyceryl-3-phosphorylcholine (2-lyso-PAF) generated from 1-0-alkyl-2-acyl-sn-glyceryl-3-phosphorylcholine by phospholipase A2 (PLA2) activity. Porins and LPS, indeed, induced PLA2-dependent mobilization of [14C]-arachidonic acid that was inhibited by p-bromodiphenacylbromide (PBDB). PBDB, an inhibitor of PLA2, also blocked PAF synthesis by preventing the mobilization of 2-lyso-PAF, the substrate for PAF-specific acetyltransferase.(ABSTRACT TRUNCATED AT 250 WORDS)
Camussi
G
G
Dipartimento di Biochimica e Biofisica, I Facoltá di Medicina e Chirurgia, Universitá di Napoli, Italy.
Biancone
L
L
Iorio
E L
EL
Silvestro
L
L
Da Col
R
R
Capasso
C
C
Rossano
F
F
Servillo
L
L
Balestrieri
C
C
Tufano
M A
MA
D001425:Bacterial Outer Membrane Proteins / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002478:Cells, Cultured; D005920:Glomerular Mesangium / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology; D010741:Phospholipases A / Q000378:metabolism; D054467:Phospholipases A2; D010972:Platelet Activating Factor / Q000096:biosynthesis*; D018272:Porins; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D001425:Bacterial Outer Membrane Proteins; D008070:Lipopolysaccharides; D010972:Platelet Activating Factor; D018272:Porins; D014409:Tumor Necrosis Factor-alpha; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium