Essential Requirement of Cytosolic Phospholipase A₂ for Stimulation of NADPH Oxidase-associated Diaphorase Activity in Granulocyte-like Cells

We have previously established a model of cytosolic phospholipase A ₂ (cPLA₂)-deficient differentiated PLB-985 cells (PLB-D cells) and demonstrated that cPLA₂-generated arachidonic acid (AA) is essential for NADPH oxidase activation. In this study we used this model to investigate the physiological role of cPLA₂ 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 (GTPγS), 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 GTPγS 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 μM 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 cPLA₂-generated AA in activation of electron transfer through the FAD reduction center of NADPH oxidase.