Salmonella typhimurium porins stimulate platelet-activating factor synthesis by human polymorphonuclear neutrophils.
Porins, a family of hydrophobic proteins located in the outer membrane of cell-wall of Gram-negative bacteria, were shown to stimulate the synthesis and release of platelet-activating factor (PAF), a 1-O-alkyl-2-acetyl-sn-glycerol-3-phosphorylcholine mediator of inflammation and endotoxic shock produced by polymorphonuclear neutrophils. PAF synthesis was independent either from contamination by LPS or generation of TNF. Experiments with labeled precursors demonstrated that PAF was synthesized via the remodeling pathway that involves acetylation of 1-O-alkyl-sn-glyceryl-3-phosphorylcholine generated from 1-O-alkyl-2-acyl-sn-glyceryl-3-phosphorylcholine by phospholipase A2 (PLA2) activity. Porins, indeed, induced a sustained PLA2-dependent mobilization of [14C]arachidonic acid that was inhibited by p-bromodiphenacylbromide. p-Bromodiphenacylbromide, an inhibitor of PLA2, also blocked PAF synthesis by preventing the mobilization of 2-lyso-PAF, the substrate for PAF-specific acetyltransferase. The addition of 2-lyso-PAF restored PAF synthesis. The activity of acetyl CoA:2-lyso-PAF acetyltransferase was transiently increased in porin-stimulated PMN and the [3H]acetyl group was incorporated in the synthetized PAF after cell preincubation with [3H]acetyl CoA. The activation of PAF synthesis by porins as well as its release were dependent on extracellular Ca2+. Porins by forming trans-membrane channels determined a sustained influx of 45Ca2+ into the cytosol. As shown by inhibitors of Ca(2+)-calmodulin complexes, calmodulin mediated the Ca(2+)-dependent activation of enzymes involved in PAF synthesis.
Tufano
M A
MA
Istituto di Microbiologia, I Facoltà di Medicina e Chirurgia, Università di Napoli, Italy.
Tetta
C
C
Biancone
L
L
Iorio
E L
EL
Baroni
A
A
Giovane
A
A
Camussi
G
G
D016718:Arachidonic Acid / Q000378:metabolism; D001425:Bacterial Outer Membrane Proteins / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D005110:Extracellular Space / Q000502:physiology; D006801:Humans; D066298:In Vitro Techniques; D009504:Neutrophils / Q000378:metabolism*; D010972:Platelet Activating Factor / Q000096:biosynthesis*; D018272:Porins; D012486:Salmonella typhimurium / Q000276:immunology*; D013997:Time Factors
D001425:Bacterial Outer Membrane Proteins; D010972:Platelet Activating Factor; D018272:Porins; D016718:Arachidonic Acid; D002118:Calcium