Regulation of Phospholipase A₂ Activity in Undifferentiated and Neutrophil-hke HL60 Cells: Linkage Between Impaired Responses to Agonists and Absence of Protein Kinase C-dependent Phosphorylation of Cytosolic Phospholipase A₂

We compared the regulation of cytosolic phospholipase A₂ (cPLA₂) activity in undifferentiated and neutrophil-like HL60 cells. Although Ca²⁺-mobilizing P₂-purinergic receptors are expressed in both cell types, arachidonic acid (AA) release stimulated by P₂-purinergic agonists was 5-7-fold higher in the differentiated cells. Similarly, the stimulation of AA release by AlF^-₄ in intact cells or by ATP and guanosine 5′-3-O-(thio)triphosphate (GTPγS) in electropermeabilized cells was significantly higher in the differentiated cells. Treatment with phorbol 12-myristate 13-acetate (PMA) enhanced A23187-stimulated AA release in intact HL60 granulocytes with minimal effects in the undifferentiated cells. Immunoblotting experiments showed similar levels of cPLA₂ and of agonist-mediated activation of mitogen-activated protein kinase in both cell types. Experiments measuring stimulation of AA release by either melittin, using endogenously labeled intact cells, or Ca²⁺, using homogenates and exogenous substrate, indicated that undifferentiated cells do not lack an activatable PLA₂. The stimulatory effects of GTPγS and Ca²⁺ on AA release in homogenates from endogenously labeled cells suggested that undifferentiated cells display G protein-cPLA₂ coupling. Basal and PMA-stimulated phosphorylation of cPLA₂ was detected in differentiated, but not in undifferentiated cells. However, the two cell types displayed only subtle differences in the time courses of phosphorylation of mitogen-activated protein kinase triggered by agonists and PMA. The observed defect in cPLA₂ phosphorylation may represent the alteration preventing agonist-mediated stimulation of AA release in undifferentiated HL60 cells.