EFFECTS OF BREFELDIN-A ON PHOSPHATIDYLCHOLINE PHOSPHOLIPASE-D AND INOSITOLPHOSPHOLIPID METABOLISM IN HL-60 CELLS

The involvement of the small GTP-binding protein ADP-ribosylation fact or (ARF) in guanosine 5'-[gamma-thio]triphosphate (GTP[S])-dependent a ctivation of phospholipase D (PLD) in HL-60 cells has been well establ ished in vitro. In this study, we tested the effect of brefeldin A, wh ich 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 t he activation of PLD at a time (1 h) when it eliminated the activity o f the trans-Golgi enzyme galactosyltransferase. It also did not inhibi t 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 b y formyl-Met-Leu-Phe and partly inhibited (approximate to 50%) the act ivation by PMA. In contrast, long-term brefeldin A treatment did not i nhibit the effect of GTP[S] on PLD in permeabilized HL-60 cells. Long- term brefeldin A treatment completely inhibited inositol phosphate pro duction 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 Golg i function is not associated with inhibition of receptor-mediated or P MA-mediated PLD activation in HL-60 cells. However, longer-term effect s, presumably arising from the disruption of the Golgi, lead to a tota l inhibition of agonist activation of PLD and inositolphospholipid-spe cific phospholipase C. In summary, these results do not support a role for brefeldin-A-sensitive ARF in agonist regulation of PLD in HL-60 c ells.