Phospholipase D activation by P_2Z-purinoceptor agonists in human lymphocytes is dependent on bivalent cation influx Available to Purchase

The role of bivalent cations in ATP-stimulated phospholipase D (PLD) activity was investigated in human leukaemic lymphocytes. Cells were labelled with [³H]oleic acid and incubated with extracellular ATP or benzoylbenzoic ATP in the presence of 1 mM Ca²⁺ and butanol, and PLD activity was assayed by the accumulation of [³H]phosphatidylbutanol ([³H]PBut). ATP stimulated PLD activity in a dose-dependent manner, and the inhibitory effects of suramin, oxidized ATP and extracellular Mg²⁺ suggested that the effect of ATP was mediated by P_2Z purinoceptors known to be present on lymphocytes. Thapsigargin increased cytosolic [Ca²⁺] but did not stimulate PLD activity, whereas preloading cells with a Ca²⁺ chelator reduced cytosolic [Ca²⁺] and, paradoxically, potentiated ATP-stimulated [³H]PBut accumulation. ATP-stimulated [³H]PBut formation was supported by both Ba²⁺ and Sr²⁺ when they were substituted for extracellular Ca²⁺. Addition of EGTA to block bivalent cation influx inhibited the majority of ATP-stimulated PLD activity. Furthermore ATP-stimulated PLD activity showed a linear relationship to extracellular [Ba²⁺], and ATP-induced ¹³³Ba²⁺ influx also had a linear dependence on extracellular [Ba²⁺]. These results suggest that ATP stimulates PLD activity in direct proportion to the influx of bivalent cations through the P_2z-purinoceptor ion channel and that this PLD activity is insensitive to changes in bulk cytosolic [Ca²⁺].