Phosphorylation-dependent regulation of phospholipase A₂ by G-proteins and Ca²⁺ in HL60 granulocytes

We studied the regulation of arachidonic acid (AA) release by guanosine 5′-O-(3-thiotriphosphate (GTPγS) and Ca²⁺ in electropermeabilized HL60 granulocytes. Stimulation of AA release by GTPγS and Ca²⁺ was mediated by phospholipase A₂ (PLA₂) and required the presence of MgATP (EC₅₀: 100-250 μM). The nucleotide effects were Ca²⁺-dependent (maximal effects detected at 1 MM free cation). UTP and ATPγS, which stimulate AA release in intact HL60 granulocytes with potencies and efficacies similar to those of ATP, were ineffective in supporting the effects of GTPγS in electropermeabilized cells. Pretreatment with pertussis toxin affected stimulation of AA release by ATP in intact cell, without altering the nucleotide effects in permeabilized cells. We observed the protein kinase C-dependent phosphorylation of PLA₂ in permeabilized HL60 granulocytes, together with a correlation between the effects of phorbol esters and staurosporine on this reaction and on AA release. ATP-independent activation of PLA₂ by GTPγS and/or Ca²⁺ was measured in subcellular fractions prepared from HL60 granulocytes. These data appear consistent with a model in which PLA₂ activity in resting HL60 granulocytes is subjected to an inhibitory constraint that prevents its activation by Ca²⁺ and G-proteins. Removal of this constraint, either by the protein kinase C-dependent phosphorylation of the enzyme in vivo or physical disruption of the regulatory assembly (e.g. by N₂ cavitation), allows its activation by Ca²⁺ and G-proteins.