Degradation of arachidonyl phospholipids catalyzed by two phospholipases A2 and phospholipase C in a lipopolysaccharide-treated macrophage cell line RAW264.7.
The release of arachidonate was stimulated by lipopolysaccharides (LPS) from phosphatidylinositol (PI), phosphatidylcholine (PC), and phosphatidylethanolamine (PE) in a murine macrophage-like cell line, RAW264.7. We measured phospholipase activities in cell-free homogenates of macrophages with 2-arachidonyl PC, PE, and PI as substrates. The activities of two phospholipases A2, catalyzing cleavage of arachidonate preferentially either from PC or PE, were detected. These two phospholipase A2 activities showed different pH optima and Ca2+ requirements; the cleavage of arachidonate from PC showed an optimal pH of 7.0 and was Ca2+-dependent, while that from PE showed an optimal pH of 7.5 but was Ca2+-independent. The cleavage of arachidonate from PI showed a different pH profile and was Ca2+-dependent, and diglyceride (DG) was detected as well as arachidonate, suggesting that both phospholipase C and DG lipase participate in this reaction. We next examined these phospholipase activities in homogenates of macrophages pretreated with LPS. All of the phospholipase activities increased at 0.5 h after LPS treatment, and this level was retained for more than 2 h in 2-arachidonyl PC degradation, continued up to 1 h and then dropped to the control level in 2-arachidonyl PE degradation, and suddenly dropped to the control level after 0.5 h in 2-arachidonyl PI degradation. These results suggest that the cleavage of 2-arachidonate from PC, PE, and PI is essentially catalyzed through different pathways, two phospholipase A2 activities being involved in PC and PE breakdown, and phospholipase C and DG lipase activities in PI breakdown, and that the activities of these substrate-specific phospholipases change in response to LPS treatment in macrophages.
Tanaka
Y
Y
Department of Chemistry, National Institute of Health, Tokyo, Japan.
Amano
F
F
Kishi
H
H
Nishijima
M
M
Akamatsu
Y
Y
D000818:Animals; D016718:Arachidonic Acid; D001095:Arachidonic Acids / Q000378:metabolism*; D002118:Calcium / Q000494:pharmacology; D002460:Cell Line; D004492:Edetic Acid / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008071:Lipoprotein Lipase / Q000378:metabolism; D008264:Macrophages / Q000201:enzymology*; D051379:Mice; D010713:Phosphatidylcholines / Q000378:metabolism; D010714:Phosphatidylethanolamines / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism; D010740:Phospholipases / Q000378:metabolism*; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D010743:Phospholipids / Q000378:metabolism*; D010738:Type C Phospholipases / Q000378:metabolism*
D001095:Arachidonic Acids; D008070:Lipopolysaccharides; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D010716:Phosphatidylinositols; D010743:Phospholipids; D016718:Arachidonic Acid; D004492:Edetic Acid; D010740:Phospholipases; D010741:Phospholipases A; D008071:Lipoprotein Lipase; D054467:Phospholipases A2; D010738:Type C Phospholipases; D002118:Calcium