Phospholipase D activation by P2Z-purinoceptor agonists in human lymphocytes is dependent on bivalent cation influx.
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 P2Z 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 P2Z-purinoceptor ion channel and that this PLD activity is insensitive to changes in bulk cytosolic [Ca2+].
Gargett
C E
CE
Department of Haematology, Austin and Repatriation Medical Centre, Heidelberg, Vic., Australia.
Cornish
E J
EJ
Wiley
J S
JS
D000255:Adenosine Triphosphate / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D001692:Biological Transport; D002413:Cations, Divalent / Q000378:metabolism*; D004789:Enzyme Activation; D006801:Humans; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000201:enzymology / Q000378:metabolism; D008214:Lymphocytes / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D010739:Phospholipase D / Q000378:metabolism*; D058913:Purinergic Agonists
D002413:Cations, Divalent; D058913:Purinergic Agonists; D000255:Adenosine Triphosphate; D010739:Phospholipase D