Endogenous suppression of neutral-active and calcium-dependent phospholipase A2 in human polymorphonuclear leukocytes.
Phospholipase A2 activity was measured in homogenized and acid-extracted human polymorphonuclear leukocytes using [1-14C]oleate-labelled autoclaved Escherichia coli as substrate. In whole homogenate and in the supernatant and particular fractions separated by centrifugation at 150,000 X g, phospholipase activity was barely detectable (1-4 pmol/h per 10(6) cell equivalents). By contrast, acid extracts of these fractions contained over 10-times as much phospholipase activity in the dialyzed supernatants (20-300 pmol/h per 10(6) cell equivalents), whereas phospholipase inhibitor(s) were found in the sediment. The acid-solubilized phospholipase A2 activity was absolutely Ca2+-dependent and optimal at pH 7.0-7.5 with 1.0 mM added Ca2+. Addition of the resuspended sediment of the acid extract dose-dependently suppressed phospholipase activity in the supernatant; less than equivalent amounts were sufficient to inhibit 95%. Suppressor activity was lipid-extractable. After thin layer chromatography of lipid extracts, the bulk of inhibitory activity was recovered from the free fatty acid region. Analysis of the fatty acids by gas liquid chromatography showed that 63% were unsaturated. All unsaturated fatty acids tested were potent inhibitors of phospholipase A2 activity (IC50 3-10 microM). Oleoyl-CoA, hydroxyeicosatetraenoic acids and leukotriene D4 were also inhibitory, while methyl oleate, saturated fatty acids and the prostaglandins E2 and F2 alpha had no effect. These in vitro data indicate that neutral-active and calcium-dependent phospholipase A2 in human polymorphonuclear leukocytes is largely suppressed by endogenous inhibitors and suggest that unsaturated fatty acids and some of their metabolites may partly account for this suppressor activity.
D002118:Calcium / Q000502:physiology; D002855:Chromatography, Thin Layer; D004789:Enzyme Activation; D005231:Fatty Acids, Unsaturated / Q000502:physiology; D006801:Humans; D009504:Neutrophils / Q000201:enzymology*; D010740:Phospholipases / Q000097:blood*; D010741:Phospholipases A / Q000037:antagonists & inhibitors / Q000097:blood*; D054467:Phospholipases A2
D005231:Fatty Acids, Unsaturated; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium