Regulation of phospholipase A2 activity in undifferentiated and neutrophil-like 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 Ca(2+)-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 AlF4- in intact cells or by ATP and guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) 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 GTP gamma S 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.
Xing
M
M
Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106.
Wilkins
P L
PL
McConnell
B K
BK
Mattera
R
R
D000255:Adenosine Triphosphate / Q000494:pharmacology; D017607:Aluminum Compounds / Q000494:pharmacology; D016718:Arachidonic Acid / Q000378:metabolism; D015153:Blotting, Western; D003994:Bucladesine / Q000494:pharmacology; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D002454:Cell Differentiation; D002460:Cell Line; D003600:Cytosol / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D005459:Fluorides / Q000494:pharmacology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D015473:Leukemia, Promyelocytic, Acute; D009504:Neutrophils / Q000166:cytology / Q000201:enzymology*; D010741:Phospholipases A / Q000302:isolation & purification / Q000378:metabolism*; D054467:Phospholipases A2; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D017607:Aluminum Compounds; C050992:tetrafluoroaluminate; D016718:Arachidonic Acid; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000001:Calcimycin; D003994:Bucladesine; D000255:Adenosine Triphosphate; D010741:Phospholipases A; D054467:Phospholipases A2; D013755:Tetradecanoylphorbol Acetate; D005459:Fluorides; D002118:Calcium