PHOSPHOLIPASE-D ACTIVATION BY P-2Z-PURINOCEPTOR AGONISTS IN HUMAN-LYMPHOCYTES IS DEPENDENT ON BIVALENT CATION INFLUX

The role of bivalent cations in ATP-stimulated phospholipase D (PLD) a ctivity was investigated in human leukaemic lymphocytes. Cells were la belled with [H-3]oleic acid and incubated with extracellular ATP or be nzoylbenzoic ATP in the presence of 1 mM Ca2+ and butanol, and PLD act ivity was assayed by the accumulation of [H-3]phosphatidylbutanol ([H- 3]PBut). ATP stimulated PLD activity in a dose-dependent manner, and t he inhibitory effects of suramin, oxidized ATP and extracellular Mg2suggested that the effect of ATP was mediated by P-2Z purinoceptors kn own to be present on lymphocytes. Thapsigargin increased cytosolic [Ca 2+] but did not stimulate PLD activity, whereas preloading cells with a Ca2+ chelator reduced cytosolic [Ca2+] and, paradoxically, potentiat ed ATP-stimulated [H-3]PBut accumulation. ATP-stimulated [H-3]PBut for mation was supported by both Ba2+ and Sr2+ when they were substituted for extracellular Ca2+. Addition of EGTA to block bivalent cation infl ux inhibited the majority of ATP-stimulated PLD activity. Furthermore ATP-stimulated PLD activity showed a linear relationship to extracellu lar [Ba2+], and ATP-induced Ba-133(2+) influx also had a linear depend ence on extracellular [Ba2+]. These results suggest that ATP stimulate s PLD activity in direct proportion to the influx of bivalent cations through the P-2z-purinoceptor ion channel and that this PLD activity i s insensitive to changes in bulk cytosolic [Ca2+].