Abstract
We have previously established a model of cytosolic phospholipase A(2) (cPLA(2))-deficient differentiated PLB-985 cells (PLB-D cells) and demonstrated that cPLA(2)-generated arachidonic acid (AA) is essential for NADPH oxidase activation. In this study we used this model to investigate the physiological role of cPLA(2) in regulation of NADPH oxidase-associated diaphorase activity. A novel diaphorase activity assay, using 4-iodonitrotetrazolium violet as an electron acceptor, was used in permeabilized neutrophils and PLB-985 cells differentiated toward the granulocytic or monocytic phenotypes. Phorbol 12-myristate 13-acetate, guanosine 5'-3-O- (thio)triphosphate (GTP gamma S), or FMLP stimulated a similar diphenylene iodonium-sensitive diaphorase activity pattern in neutrophils and in differentiated parent PLB-985 cells. This diaphorase activity was not detected in undifferentiated cells, but developed during differentiation. Furthermore, diaphorase activity could not be stimulated in permeabilized neutrophils from X-linked CGD patients and in differentiated gp91(phox)-targeted PLB-985 cells that lacked normal expression of gp91(phox), but was restored to these cells following transduction with retrovirus encoding gp91(phox). The differentiated PLB-D cells showed no diaphorase activity when stimulated by either GTP gamma S or FMLP, and only partial activation when stimulated with phorbol 12-myristate 13-acetate. Diaphorase activity in response to either agonists was fully restored by the addition of 10 microm free AA. The permeabilized cell 4-iodonitrotetrazolium violet reduction assay offers a unique tool for the evaluation of NADPH oxidase-associated diaphorase activity in stimulated whole cells. These results establish an essential and specific physiological requirement of cPLA(2)-generated AA in activation of electron transfer through the FAD reduction center of NADPH oxidase.
Highlights
From the ‡Infectious Diseases Laboratory, Department of Clinical Biochemistry, Faculty of Health Sciences, Ben-Gurion University of the Negev and Soroka Medical Center, Beer Sheva 84105, Israel and the §Laboratory of Host Defenses, NIAID, National Institutes of Health, Bethesda, Maryland 20892-1886
We have previously established a model of cytosolic phospholipase A2-deficient differentiated PLB-985 cells (PLB-D cells) and demonstrated that cPLA2-generated arachidonic acid (AA) is essential for activation of NADPH oxidase through undefined mechanisms [19]
The iodonitrotetrazolium violet (INT) reduction assay was established in permeabilized human neutrophils (Table I) and applied to permeabilized PLB-985 cells differentiated toward a granulocytic phenotype (Table II)
Summary
Vol 276, No 36, Issue of September 7, pp. 33495–33503, 2001 Printed in U.S.A. Essential Requirement of Cytosolic Phospholipase A2 for Stimulation of NADPH Oxidase-associated Diaphorase Activity in Granulocyte-like Cells*. The permeabilized cell 4-iodonitrotetrazolium violet reduction assay offers a unique tool for the evaluation of NADPH oxidase-associated diaphorase activity in stimulated whole cells These results establish an essential and specific physiological requirement of cPLA2-generated AA in activation of electron transfer through the FAD reduction center of NADPH oxidase. Stimulation results in translocation of cytosolic NADPH oxidase components to the membrane, where they interact with the flavocytochrome to form the activated oxidase This activated enzyme transfers electrons from cytosolic NADPH to extracellular or phagosomal molecular oxygen through the electron transport chain. Cross et al [5, 15,16,17,18] have recently demonstrated this novel NADPH oxidase-associated diaphorase activity, present in the membrane fraction of activated neutrophils, using the artificial electron acceptor iodonitrotetrazolium violet (INT), which interacts directly with the reduced flavin center of the flavocytochrome.
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