Phosphatidylinositol hydrolysis by phospholipase A2 and C activities in human peripheral blood neutrophils.
We describe here and partially characterize a Ca(2+)-independent phospholipase A2 that acts on phosphatidylinositol in normal human peripheral blood neutrophils. Neutrophils incubated with myo-[3H]inositol to form [3H]phosphatidylinositol and then stimulated with the calcium ionophore A23187 produced [3H]lysophosphatidylinositol. This deacylation was further characterized in cell sonicates by the specific release of [3H]arachidonic acid from exogenous [1-14C]stearoyl-2-[3H]arachidonyl-phosphatidylinositol. This phospholipase A2 is Ca2+ independent, retaining full activity in the presence of 10 mM EDTA, and is optimally active at alkaline pH (pH 9). A phosphatidylinositol-hydrolyzing phospholipase C activity was characterized by the production of [3H]-/[14C]-diglycerides. This phospholipase C activity is dependent on the presence of exogenous Ca2+ and is optimally active at neutral pH (pH 7.5). The lipoxygenase/cyclooxygenase inhibitors eicosatetraenoic acid and nordihydroguaiaretic acid and the calmodulin antagonist trifluoperazine were the only compounds tested that showed significant inhibition of phospholipase A2 activity. However, none of these phosphatidylinositol-hydrolyzing phospholipase A2 inhibitory compounds resulted in the accumulation of any radiolabeled diglyceride, monoglyceride, or phosphatidic acid intermediates. Following subcellular fractionation on sucrose density gradients, it was found that the plasma membrane-enriched fractions contained the highest specific activity for phospholipase A2; however, the cytosolic fraction contained a large part of the total phospholipase A2 activity. Furthermore, when neutrophils were first exposed to several agents, including lipopolysaccharide, phorbol myristate acetate, or N-formyl-methionyl-leucyl- phenylalanine, and then subfractionated, there was a significant translocation of the enzyme activity from the cytosolic fraction to the membrane-enriched fractions. These data suggest that this Ca(2+)-independent, phosphatidylinositol-hydrolyzing phospholipase A2 may play an important role in early cell activation, providing free arachidonic acid for subsequent metabolism into biologically active eicosanoids.
Smith
D M
DM
Department of Biochemistry, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, North Carolina.
Waite
M
M
D016718:Arachidonic Acid / Q000097:blood; D000001:Calcimycin / Q000494:pharmacology; D002122:Calcium Chloride / Q000494:pharmacology; D002462:Cell Membrane / Q000201:enzymology; D002855:Chromatography, Thin Layer; D003600:Cytosol / Q000201:enzymology; D003840:Deoxycholic Acid / Q000494:pharmacology; D003902:Detergents / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008070:Lipopolysaccharides / Q000494:pharmacology; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology*; D017830:Octoxynol; D010716:Phosphatidylinositols / Q000097:blood* / Q000302:isolation & purification; D010741:Phospholipases A / Q000097:blood*; D054467:Phospholipases A2; D011092:Polyethylene Glycols / Q000494:pharmacology; D013229:Stearic Acids / Q000097:blood; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D010738:Type C Phospholipases / Q000097:blood*
D003902:Detergents; D008070:Lipopolysaccharides; D010716:Phosphatidylinositols; D013229:Stearic Acids; D003840:Deoxycholic Acid; D016718:Arachidonic Acid; D000001:Calcimycin; D011092:Polyethylene Glycols; C031183:stearic acid; D009240:N-Formylmethionine Leucyl-Phenylalanine; D017830:Octoxynol; D004492:Edetic Acid; D010741:Phospholipases A; D054467:Phospholipases A2; D010738:Type C Phospholipases; D002122:Calcium Chloride; D013755:Tetradecanoylphorbol Acetate