Activation of cytosolic phospholipase A(2) in permeabilized human neutrophils

Neutrophils (PMN) contain two types of phospholipase A(2) (PLA(2)), a 14 kDa 'secretory' Type II PLA(2) (sPLA(2)) and an 85 kDa 'cytosolic' PLA(2) (cPLA(2)), that differ in a number of key characteristics: (1) cPLA(2), prefers arachidonate (AA) as a substrate but hydrolyzes all phospholipids; sPLA(2) is not AA specific but prefers ethanolamine containing phosphoacylglycerols. (2) cPLA(2) is active at nM calcium (Ca2+) concentrations; sPLA(2) requires mu M Ca2+ levels. (3) cPLA(2) activity is regulated by phosphorylation; sPLA(2) lacks phosphorylation sites. (4) cPLA(2) is insensitive to reduction; sPLA(2) 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 PLA(2) were activated in porated cells under conditions designed to differentiate between the two enzymes. PMN were labeled with [H-3]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. [H-3]AA and AA mass release occurred in parallel over time. A hydrolyzable form of ATP was necessary for maximum AA release and staurosporin inhibited PLA(2) activation. Dithiothreitol treatment had little affect on [H-3]AA release and metabolism but inhibited AA mass release. Assay of cell supernatants after cofactor addition did not detect sPLA(2) activity and the cytosolic buffer utilized did not support activity of recombinant sPLA(2). These results strongly suggested that cPLA(2) 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 PLA(2) in PMN.