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 p hospholipids; sPLA(2) is not AA specific but prefers ethanolamine cont aining phosphoacylglycerols. (2) cPLA(2) is active at nM calcium (Ca2) concentrations; sPLA(2) requires mu M Ca2+ levels. (3) cPLA(2) activ ity is regulated by phosphorylation; sPLA(2) lacks phosphorylation sit es. (4) cPLA(2) is insensitive to reduction; sPLA(2) is inactivated by agents that reduce disulfide bonds. We utilized PMN permeabilized wit h Staphylococcus aureus alpha-toxin to determine whether one or both f orms of PLA(2) were activated in porated cells under conditions design ed to differentiate between the two enzymes. PMN were labeled with [H- 3]AA to measure release from phosphatidylcholine and phosphatidylinosi tol; gas chromatography-mass spectrometry was utilized to determine to tal AA release (mainly from phosphatidylethanolamine) and to assess ol eate and linoleate mass. A combination of 500 nM Ca2+, a guanine nucle otide, 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 oc curred 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 meta bolism but inhibited AA mass release. Assay of cell supernatants after cofactor addition did not detect sPLA(2) activity and the cytosolic b uffer utilized did not support activity of recombinant sPLA(2). These results strongly suggested that cPLA(2) was the enzyme activated in th e permeabilized cell model and this is the first report which unambigu ously demonstrates AA release in response to activation of a specific type of PLA(2) in PMN.