title: Effects of brefeldin A on phosphatidylcholine phospholipase D and inositolphospholipid metabolism in HL-60 cells.
abstract: The involvement of the small GTP-binding protein ADP-ribosylation factor (ARF) in guanosine 5'-[gamma-thio]triphosphate (GTP[S])-dependent activation of phospholipase D (PLD) in HL-60 cells has been well established in vitro. In this study, we tested the effect of brefeldin A, which prevents ARF activation by inhibiting guanine-nucleotide-exchange activity, on PLD stimulation by receptor agonists (formyl-Met-Leu-Phe and ATP) and by the phorbol ester phorbol 12-myristate 13-acetate (PMA) in differentiated HL-60 cells. However, brefeldin A did not affect the activation of PLD at a time (1 h) when it eliminated the activity of the trans-Golgi enzyme galactosyltransferase. It also did not inhibit PLD activity in Golgi-enriched membranes treated with GTP[S] with or without ARF in vitro. However, longer times of brefeldin A treatment (>6 h), progressively and completely inhibited the activation of PLD by formyl-Met-Leu-Phe and partly inhibited (approximately 50%) the activation by PMA. In contrast, long-term brefeldin A treatment did not inhibit the effect of GTP[S] on PLD in permeabilized HL-60 cells. Long-term brefeldin A treatment completely inhibited inositol phosphate production in response to formyl-Met-Leu-Phe and ATP, indicating that it affected inositolphospholipid-specific phospholipase C activity. These data indicate that the rapid inhibitory effect of brefeldin A on Golgi function is not associated with inhibition of receptor-mediated or PMA-mediated PLD activation in HL-60 cells. However, longer-term effects, presumably arising from the disruption of the Golgi, lead to a total inhibition of agonist activation of PLD and inositolphospholipid-specific phospholipase C. In summary, these results do not support a role for brefeldin-A-sensitive ARF in agonist regulation of PLD in HL-60 cells.
authors.affiliation: Howard Hughes Medical Institute and Vanderbilt University School of Medicine, Department of Molecular Physiology and Biophysics, Nashville, TN 37232-0295, USA.
authors.forename: I
authors.initials: I
authors.lastname: Guillemain
authors.forename: J H
authors.initials: JH
authors.lastname: Exton
mesh_terms: D020727:ADP-Ribosylation Factors; D000255:Adenosine Triphosphate / Q000494:pharmacology; D020126:Brefeldin A; D003517:Cyclopentanes / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism; D005700:Galactosyltransferases / Q000378:metabolism; D006056:Golgi Apparatus / Q000187:drug effects / Q000201:enzymology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D018922:HL-60 Cells; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism; D007700:Kinetics; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D010713:Phosphatidylcholines / Q000378:metabolism*; D010716:Phosphatidylinositols / Q000378:metabolism*; D010739:Phospholipase D / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D010738:Type C Phospholipases / Q000378:metabolism*
chemical_list: D003517:Cyclopentanes; D004791:Enzyme Inhibitors; D007295:Inositol Phosphates; D010713:Phosphatidylcholines; D010716:Phosphatidylinositols; D020126:Brefeldin A; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D009240:N-Formylmethionine Leucyl-Phenylalanine; D000255:Adenosine Triphosphate; D005700:Galactosyltransferases; D010738:Type C Phospholipases; D010739:Phospholipase D; D019204:GTP-Binding Proteins; D020727:ADP-Ribosylation Factors; D013755:Tetradecanoylphorbol Acetate