Essential requirement of cytosolic phospholipase A(2) 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 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.
Laboratory of Infectious Diseases, Department of Clinical Biochemistry, Faculty of Health Sciences, Ben-Gurion University of the Negev and Soroka Medical Center, Beer-Sheva 84105, Israel.
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Lowenthal
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Levy
D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D000889:Anti-Arrhythmia Agents / Q000494:pharmacology; D002117:Calcitriol / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D003600:Cytosol / Q000201:enzymology; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009504:Neutrophils / Q000187:drug effects / Q000502:physiology*; D010586:Phagocytes / Q000502:physiology; D010741:Phospholipases A / Q000378:metabolism*; D017494:Proton Pumps / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection; D015032:Zinc / Q000494:pharmacology
D000889:Anti-Arrhythmia Agents; D017494:Proton Pumps; D011994:Recombinant Proteins; D017923:Sodium-Hydrogen Exchangers; D013481:Superoxides; D000584:Amiloride; D010741:Phospholipases A; D002117:Calcitriol; D015032:Zinc; D013755:Tetradecanoylphorbol Acetate; C039614:ethylisopropylamiloride; D004121:Dimethyl Sulfoxide