EVIDENCE THAT SECRETORY PHOSPHOLIPASE A(2), PLAYS A ROLE IN ARACHIDONIC-ACID RELEASE AND EICOSANOID BIOSYNTHESIS BY MAST-CELLS

This study investigated the role of secretory PLA(2) (sPLA(2)) in arachidonic acid (AA) release and lipid mediator generation in mouse bone marrow-derived mast cells. Initial studies indicated that mast cells contain multiple PLA(2) activities and secreted PLA(2) activity upon Ag stimulation. The secreted PLA(2) activity is blocked by DTT and by a group II PLA(2)-neutralizing Ab. Mast cells incubated with groups I or II PLA(2) selectively release polyunsaturated fatty acids, such as AA, into supernatant fluids. The bulk of AA released by sPLA(2) is derived from phosphatidylethanolamine. The fatty acids released by extracellular PLA(2) mimic those found in supernatant fluids after Ag stimulation of mast cells. Incubation of mast cells with PLA(2) generates cyclooxygenase (CO) products but no 5-lipoxygenase (5-LO) products. Ag, but not PLA(2), induces the translocation of 5-LO to cellular membranes and the formation of 5-LO products. The addition of sPLA(2) to Ag-stimulated mast cells increases the synthesis of 5-LO products. Octadeuterated AA ((2)H(8)AA) added to the outside of cells to trace extracellular AA metabolism is rapidly converted to CO products. Addition of sPLA(2), or Ag in combination with (2)H(8)AA reduces the quantity of (2)H(8)AA converted to deuterated CO products, when compared with adding (2)H(8)AA alone. The reduction of deuterated CO products can be accounted for with increases in nondeuterated CO products. (2)H(8)AA is only converted to 5-LO products when mast cells are activated with Ag. The amount of (2)H(8)AA converted to deuterated 5-LO products is reduced by the addition of PLA(2) to Ag-stimulated cells. The competitive formation of deuterated and nondeuterated products observed when mast cells are incubated with (2)H(8)AA, indicates that extracellular AA released by sPLA(2) is used for both CO and 5-LO product formation. Taken together, these data reveal a role for sPLA(2) in the release of AA and demonstrate that AA released by this mechanism is used for eicosanoid generation.