Abstract
The NADPH oxidase-producing superoxide is the major mechanism by which phagocytes kill invading pathogens. We 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 (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 cPLA(2) 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% Me(2)SO or 5 x 10(-8) M 1, 25-dihydroxyvitamin D(3). Activation of differentiated PLB cells resulted in a Zn(2+)-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 cPLA(2) in H(+) channel activation. The presence of the H(+) channel inhibitor Zn(2+) 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 cPLA(2) does not participate in the regulation of this antiporter. These results establish an essential and specific physiological requirement of cPLA(2)-generated AA for activation of the H(+) channel and suggest the participation of this channel in the regulation of NADPH oxidase activity.
Highlights
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 to host defense
We previously established a model of cytosolic phospholipase A2-deficient differentiated PLB-985 cells and demonstrated that cPLA2-generated arachidonic acid is essential, by an unknown mechanism, for the activation of NADPH oxidase [27]
PHi Changes Stimulated with PMA—This set of experiments was performed in order to determine whether PMA induces a change in pHi in differentiated PLB-985 cells similar to that observed in human neutrophils
Summary
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 to host defense. These two phenomena reflect the significantly higher PMA-stimulated NADPH oxidase activity in peripheral blood neutrophils generating 32 Ϯ 4.5 nmol of O2/106 cells/min compared with granulocyte-like PLB cells or monocyte-like PLB cells producing 14.53 Ϯ 2.8 and 10.85 Ϯ 3.3 nmol of O2/106 cells/min, respectively (the results are the mean Ϯ S.E. from 10 different experiments).
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