Essential requirement of cytosolic phospholipase A₂ for activation of the H⁺ channel in phagocyte-like cells

The NADPH oxidase-producing superoxide is the major mechanism by which phagocytes kill invading pathogens. We previously established a model of cytosolic phospholipase A₂ (cPLA₂)-deficient differentiated PLB985 cells (PLB-D cells) and demonstrated that cPLA₂-generated arachidonic acid (AA) is essential for NADPH oxidase activation (Dana, R., Leto, T., Malech, H., and Levy, R. (1998) J. Biol. Chem. 273, 441-445). In the present study, we used this model to determine the physiological role of cPLA₂ in the regulation of both the H⁺ channel and the Na⁺/H⁺ antiporter and to study whether NADPH oxidase activation is regulated by either of these transporters. PLB-D cells and two controls: parent PLB-985 cells and PLB-985 cells transfected with the vector only (PLB cells) were differentiated using 1.25% Me₂SO or 5 x 10^-8 M 1,25-dihydroxyvitamin D₃. Activation of differentiated PLB cells resulted in a Zn²⁺sensitive alkalization, indicating H⁺ channel activity. In contrast, differentiated PLB-D cells failed to activate the H⁺ channel, but the addition of exogenous AA fully restored this activity, indicating the role of cPLA₂ in H⁺ channel activation. The presence of the H⁺ channel inhibitor Zn²⁺ caused significant inhibition of NADPH oxidase activity, suggesting a role of the H⁺ channel in regulating oxidase activity. Na⁺/H⁺ antiporter activity was stimulated in differentiated PLB-D cells, indicating that cPLA₂ does not participate in the regulation of this antiporter. These results establish an essential and specific physiological requirement of cPLA₂-generated AA for activation of the H⁺ channel and suggest the participation of this channel in the regulation of NADPH oxidase activity.