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 A(2) (cPLA(2)) 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 cPLA(2) (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 cPLA(2), 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 cPLA(2); however, addition of AA restored the efflux of protons in these cells. These results establish an essential and specific physiological requirement of cPLA(2)-generated AA in the activation of proton flux through the NT fragment of gp91(phox).
Mankelow
Tosti J
TJ
Department of Biochemistry, School of Medical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, UK.
Pessach
Etai
E
Levy
Rachel
R
Henderson
Lydia M
LM
D002104:Cadmium / Q000494:pharmacology; D002460:Cell Line; D003600:Cytosol / Q000187:drug effects / Q000201:enzymology*; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D008562:Membrane Glycoproteins / Q000378:metabolism*; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000378:metabolism*; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology; D010446:Peptide Fragments / Q000502:physiology*; D010586:Phagocytes / Q000187:drug effects / Q000201:enzymology; D010741:Phospholipases A / Q000172:deficiency / Q000502:physiology*; D054467:Phospholipases A2; D017494:Proton Pumps / Q000378:metabolism*; D011522:Protons; D013481:Superoxides / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D008562:Membrane Glycoproteins; D010446:Peptide Fragments; D017494:Proton Pumps; D011522:Protons; D002104:Cadmium; D013481:Superoxides; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; D010741:Phospholipases A; D054467:Phospholipases A2; D013755:Tetradecanoylphorbol Acetate