Essential Requirement of Cytosolic Phospholipase A2for Stimulation of NADPH Oxidase-associated Diaphorase Activity in Granulocyte-like Cells

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␥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␥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 M 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.The phagocyte NADPH oxidase is a multicomponent transport chain that transfers electrons from NADPH to molecular oxygen and generates superoxide, a precursor of microbicidal oxidants important for host defense. NADPH oxidase subunits include three cytoplasmic components, p47 phox , p67 phox , and Rac2, and a membrane flavocytochrome b 558 composed of gp91 phox and p22 phox (1, 2). The flavocytochrome b 558 contains three redox centers (FAD and 2 hemes) and an NADPH binding site (3). In addition, a H ϩ channel was identified within the N-terminal 230 amino acids of gp91 phox (2, 4). In phagocytes, 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. The flavin center transfers electrons from the physiological electron donor, NADPH, to two membrane-imbedded hemes of the cytochrome. The latter serves as the terminal electron donor to oxygen (2, 5). It has been demonstrated that the single electron tran...

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