Role of phospholipase D in Pasteurella haemolytica leukotoxin-induced increase in phospholipase A(2) activity in bovine neutrophils

The effects of Pasteurella haemolytica leukotoxin (LKT) on the activity of phospholipase D (PLD) and the regulatory interaction between PLD and phosph olipase A(2) (PLA(2)) were investigated in assays using isolated bovine neu trophils labeled with tritiated phospholipid substrates of the two enzymes. Exposure of [H-3]lysophosphatidylcholine-labeled neutrophils to LKT caused concentration- and time-dependent production of phosphatidic acid (PA), th e product of PLD, LKT-induced generation of PA was dependent on extracellul ar calcium. Both production of PA and metabolism of [H-3]-arachidonate ([H- 3]AA)-labeled phospholipids by PLA(2) were inhibited when ethanol was used to promote the alternative PLD-mediated transphosphatidylation reaction, re sulting in the production of phosphatidylethanol rather than PA. The role o f PA in regulation of PLA(2) activity was then confirmed by means of an add -back experiment, whereby addition of PA in the presence of ethanol restore d PLA(2)-mediated release of radioactivity from neutrophil membranes. Consi dering the involvement of chemotactic phospholipase products in the pathoge nesis of pneumonic pasteurellosis, development and use of anti-inflammatory agents that inhibit LKT-induced activation of PLD and PLA(2) may improve t herapeutic management of the disease.