Characterization of phospholipase A2 release by elicited-peritoneal macrophage and its relationship to eicosanoid production.
Elicited guinea pig peritoneal macrophages released a soluble phospholipase A2 (PLA2) into conditioned media that was biochemically and pharmacologically similar to the recombinant human (rh) type II 14 kDa PLA2. The level of activity found in the 24 h media positively correlated with cell number and was reduced by actinomycin D and cycloheximide suggesting the enzyme was being constitutively synthesized. The enzyme protein accumulated in the media over 10-24 h and remained at maximal levels over 40 h as demonstrated by both activity and ELISA measurements. This was preceded by an increase in total cell-associated sn-2-acylhydrolytic activity which reached maximal levels by 12-17 h of culture and remained elevated to 40 h. Treatment of cell homogenate with dithiothreitol (DTT) revealed a rise in DTT-insensitive sn-2-acylhydrolytic activity which increased between 12 and 24 h. Prostaglandin (PG) E2 but not leukotriene (LT)B4 accumulated in the media and reached maximal levels by 24 h. This paralleled the release of secreted type II 14 kDa-like PLA2 and the rise in DTT-insensitive cell-associated activity, but not upregulation and formation of new cyclooxygenase after aspirin treatment. Overnight exposure to a non-cell permeable selective 14 kDa PLA2 inhibitor or neutralizing mAb interfered with expression of PLA2 activity but not PGE2 accumulation over 24 h. This indicated that the secreted PLA2 was not directly involved in PGE2 biosynthesis. Exposure of the elicited macrophages to the cell permeable 14 kDa PLA2 inhibitor, 12-epi-scalaradial also did not effect PGE2 accumulation. Taken together, elicited guinea-pig macrophages release 14 kDa PLA2 upon culture, but this activity appears not to be related to the concomitant accumulation in PGE2. The role of the cell-associated PLA2 activity(s) in PGE2 formation cannot be ruled out.
Marshall
L A
LA
SmithKline Beecham Pharmaceuticals, Inflammation & Respiratory Pharmacology, King of Prussia, PA 19406-0939.
Bolognese
B
B
Roshak
A
A
D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology; D002478:Cells, Cultured; D002845:Chromatography; D003470:Culture Media; D015232:Dinoprostone / Q000096:biosynthesis / Q000378:metabolism; D015777:Eicosanoids / Q000096:biosynthesis*; D004797:Enzyme-Linked Immunosorbent Assay; D005110:Extracellular Space / Q000201:enzymology; D006168:Guinea Pigs; D006801:Humans; D006863:Hydrogen-Ion Concentration; D017737:Macrophages, Peritoneal / Q000201:enzymology* / Q000378:metabolism* / Q000502:physiology; D008297:Male; D010741:Phospholipases A / Q000378:metabolism* / Q000502:physiology; D054467:Phospholipases A2; D011451:Prostaglandin-Endoperoxide Synthases / Q000096:biosynthesis; D014176:Protein Biosynthesis / Q000187:drug effects; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013997:Time Factors; D014158:Transcription, Genetic / Q000187:drug effects
D003470:Culture Media; D015777:Eicosanoids; D013439:Sulfhydryl Reagents; D016718:Arachidonic Acid; D011451:Prostaglandin-Endoperoxide Synthases; D010741:Phospholipases A; D054467:Phospholipases A2; D015232:Dinoprostone; D002118:Calcium