THE REGULATION BY PHOSPHORYLATION OF PRIMING OF PHOSPHOLIPASE A(2) ACTIVITY IN THE NEUTROPHIL MODEL SYSTEM, DIFFERENTIATED HL60 CELLS

1 Differentiated HL60 cells have been utilized as a model system to ex amine the 'priming' of neutrophil phospholipase A(2) activity. In cont rol cells activation of phospholipase A(2) by a 5 min stimulation with the chemotactic peptide formyl-methionyl-lencyl-phenylalanine (100 nM ) was essentially undetectable. When cells were primed by preincubatio n with 5 mu M cytochalasin B for 5 min arachidonate release, a measure of phospholipase A(2) activation, was observed within 20 s. 2 Priming by cytochalasin B did not involve or require a change in intracellula r free calcium concentration. 3 Priming was associated with an increas e in general protein tyrosine phosphorylation and could also be induce d by the receptor tyrosine kinase agonist granulocyte macrophage colon y-stimulating factor (GM-CSF, 20 ng ml(-1)) and be mimicked by treatme nt 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 phosph olipase A(2) 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 ass ociation of the phospholipase and this may be regulated by tyrosine ki nase activities.