The requirement of cytosolic phospholipase A2 for the PMA activation of proton efflux through the N-terminal 230-amino-acid fragment of gp91phox

The absolute requirement for the 85 kDa cytosolic phospholipase A₂ (cPLA₂) in the PMA stimulation of proton efflux through the NADPH-oxidase-associated proton channel, has previously been demonstrated using a PLB-985 cell line deficient in cPLA₂ (PLB-D). The flux of protons in Chinese-Hamster ovary (CHO) cells that express the N-terminal 230-amino-acid (NT) fragment of gp91^phox is activated by arachidonic acid (AA) added externally. To investigate the physiological role of cPLA₂, and the intracellular AA that it releases, in the activation of proton flux through the NT fragment of gp91^phox, this fragment was stably expressed in PLB-985 cells (PLB-985 NT) and in PLB-D cells (PLB-D NT). The expression of the NT fragment of gp91^phox by itself in PLB-985 did not initiate differentiation and did not alter their ability to undergo differentiation after the addition of DMSO. Addition of PMA induced a proton efflux from undifferentiated PLB-985 NT cells expressing the NT fragment of gp91^phox, which was inhibited by zinc. In contrast, PMA failed to activate proton efflux in undifferentiated PLB-D NT cells, lacking the expression of cPLA₂; however, addition of AA restored the efflux of protons in these cells. These results establish an essential and specific physiological requirement of cPLA₂-generated AA in the activation of proton flux through the NT fragment of gp91^phox.