The regulation by phosphorylation of ‘priming' of phospholipase A₂ activity in the neutrophil model system, differentiated HL60 cells

Abstract

1. Differentiated HL60 cells have been utilized as a model system to examine the ‘priming' of neutrophil phospholipase A₂ activity. In control cells activation of phospholipase A₂ by a 5 min stimulation with the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (100 nM) was essentially undetectable. When cells were primed by preincubation with 5 μM cytochalasin B for 5 min arachidonate release, a measure of phospholipase A₂ activation, was observed within 20 s.

2. Priming by cytochalasin B did not involve or require a change in intracellular free calcium concentration.

3. Priming was associated with an increase in general protein tyrosine phosphorylation and could also be induced by the receptor tyrosine kinase agonist granulocyte macrophage colony-stimulating factor (GM-CSF, 20 ng ml⁻¹) and be mimicked by treatment with the phosphotyrosine phosphatase inhibitor perhydrovanadate (0.5 mM). However, an increase in MAP kinase activity was not involved in the priming process.

4. Western blot analysis demonstrated that phospholipase A₂ was phosphorylated in both control and primed cells, but that an increase in the amount of membrane associated enzyme was found in the primed cells.

5. Thus priming appears to be due to membrane association of the phospholipase and this may be regulated by tyrosine kinase activities.

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