Activation of cytosolic phospholipase A2 in permeabilized human neutrophils.
Neutrophils (PMN) contain two types of phospholipase A2 (PLA2), a 14 kDa 'secretory' Type II PLA2 (sPLA2) and an 85 kDa 'cytosolic' PLA2 (cPLA2), that differ in a number of key characteristics: (1) cPLA2 prefers arachidonate (AA) as a substrate but hydrolyzes all phospholipids; sPLA2 is not AA specific but prefers ethanolamine containing phosphoacylglycerols. (2) cPLA2 is active at nM calcium (Ca2+) concentrations; sPLA2 requires microM Ca2+ levels. (3) cPLA2 activity is regulated by phosphorylation; sPLA2 lacks phosphorylation sites. (4) cPLA2 is insensitive to reduction; sPLA2 is inactivated by agents that reduce disulfide bonds. We utilized PMN permeabilized with Staphylococcus aureus alpha-toxin to determine whether one or both forms of PLA2 were activated in porated cells under conditions designed to differentiate between the two enzymes. PMN were labeled with [3H]AA to measure release from phosphatidylcholine and phosphatidylinositol; gas chromatography-mass spectrometry was utilized to determine total AA release (mainly from phosphatidylethanolamine) and to assess oleate and linoleate mass. A combination of 500 nM Ca2+, a guanine nucleotide, and stimulation with n-formyl-met-leu-phe (FMLP) were necessary to induce maximal AA release in permeabilized PMN measured by either method; AA was preferentially released. [3H]AA and AA mass release occurred in parallel over time. A hydrolyzable form of ATP was necessary for maximum AA release and staurosporin inhibited PLA2 activation. Dithiothreitol treatment had little affect on [3H]AA release and metabolism but inhibited AA mass release. Assay of cell supernatants after cofactor addition did not detect sPLA2 activity and the cytosolic buffer utilized did not support activity of recombinant sPLA2. These results strongly suggested that cPLA2 was the enzyme activated in the permeabilized cell model and this is the first report which unambiguously demonstrates AA release in response to activation of a specific type of PLA2 in PMN.
Bauldry
S A
SA
Bowman Gray School of Medicine of Wake Forest University, Winston-Salem, NC 27157, USA.
Wooten
R E
RE
Bass
D A
DA
D000255:Adenosine Triphosphate / Q000378:metabolism; D000470:Alkaloids / Q000494:pharmacology; D016718:Arachidonic Acid / Q000096:biosynthesis* / Q000378:metabolism; D001427:Bacterial Toxins; D002118:Calcium / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology; D004229:Dithiothreitol / Q000494:pharmacology; D004789:Enzyme Activation; D006460:Hemolysin Proteins; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism*; D019787:Linoleic Acid; D008041:Linoleic Acids / Q000096:biosynthesis; D009504:Neutrophils / Q000201:enzymology*; D019301:Oleic Acid; D009829:Oleic Acids / Q000096:biosynthesis; D010741:Phospholipases A / Q000737:chemistry / Q000378:metabolism*; D054467:Phospholipases A2; D019311:Staurosporine
D000470:Alkaloids; D001427:Bacterial Toxins; D006460:Hemolysin Proteins; D007527:Isoenzymes; D008041:Linoleic Acids; D009829:Oleic Acids; C021091:staphylococcal alpha-toxin; D016718:Arachidonic Acid; D019301:Oleic Acid; D000255:Adenosine Triphosphate; D019787:Linoleic Acid; D010741:Phospholipases A; D054467:Phospholipases A2; D019311:Staurosporine; D002118:Calcium; D004229:Dithiothreitol