Reversible activation of NADPH oxidase in membranes of HL-60 human leukemic cells

Abstract

NADPH oxidase in membranes of undifferentiated and dimethylsulphoxide-differentiated HL-60 cells was activated by arachidonic acid (AA) in the presence of Mg 2+ and a cytosolic cofactor (CF) found in differentiated HL-60 cells. Basal superoxide ( O 2 − ) formation was enhanced several-fold by addition of the stable GTP-analogue, guanosine 5′-O-(3-thiotriphosphate) (GTPγS), prior to AA and was completely prevented by that of GDP. Basal and GTPγS-stimulated O 2 − formation was terminated by GDP. In the presence of Mg 2+ or EDTA, basal O 2 − formation ceased after 25 or 10 min, respectively, and was reinitiated by GTPγS or GTPγS plus Mg 2+. Albumin terminated O 2 − formation, which was reactivated by AA in the presence of GTPγS. Our results show that (1) activation of NADPH oxidase in HL-60 membranes is dependent on endogenous GTP, Mg 2+, AA and CF, which is induced during myeloid differentiation, and that (2) NADPH oxidase activation is a reversible process modulated by exogenous guanine nucleotides at various stages of activity of NADPH oxidase. We suggest crucial roles of guanine nucleotide-binding proteins in the activation, deactivation and reactivation of the enzyme.