Activation of human neutrophil phospholipase D by three separable mechanisms.
Activation of human neutrophils by receptor-mediated agonists, the Ca2+ ionophore A23187, or the protein kinase C activator phorbol myristate acetate all stimulated phospholipase D activity. This was demonstrated by the increased formation of phosphatidic acid, and in the presence of ethanol, phosphatidylethanol (PEt) accumulation. EGTA completely inhibited A23187-induced PEt formation, but only one-half of the fMLP-induced PEt accumulation. Staurosporin, an inhibitor of protein kinase C, strongly inhibited PMA-induced PEt formation, but actually stimulated the formation of PEt in response to fMLP by several-fold. Thus, increased cytosolic Ca2+ and activated protein kinase C can each lead to activation of phospholipase D, but neither is required for receptor-mediated activation of phospholipase D activity. Wortmannin is an irreversible inhibitor of the oxidative burst, but does not inhibit NADPH oxidase or known components of signal transduction. Wortmannin inhibited activation of phospholipase D in response to fMPL. It did not directly inhibit phospholipase D, as the response to A23187 was unaffected. Wortmannin did not inhibit other fMPL-stimulated events, such as aggregation or adherence. We conclude that inhibition by wortmannin defines a third pathway to activation of phospholipase D. Further, its effect on phospholipase D correlates with its effect on the respiratory burst.
Reinhold
S L
SL
Department of Pharmacology, Nora Eccles Harrison Cardiovascular Research and Training Institute, University of Utah, Salt Lake City 84112.
Prescott
S M
SM
Zimmerman
G A
GA
McIntyre
T M
TM
D000730:Androstadienes / Q000494:pharmacology; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D002448:Cell Adhesion / Q000187:drug effects; D002449:Cell Aggregation / Q000187:drug effects; D004533:Egtazic Acid / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D020404:Glycerophospholipids; D006801:Humans; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000201:enzymology*; D010712:Phosphatidic Acids / Q000378:metabolism; D010739:Phospholipase D / Q000378:metabolism*; D010740:Phospholipases / Q000378:metabolism*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D000077191:Wortmannin
D000730:Androstadienes; D020404:Glycerophospholipids; D010712:Phosphatidic Acids; C051521:phosphatidylethanol; D000001:Calcimycin; D004533:Egtazic Acid; D009240:N-Formylmethionine Leucyl-Phenylalanine; D011493:Protein Kinase C; D010740:Phospholipases; D010739:Phospholipase D; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium; D000077191:Wortmannin