Phospholipase D activation by P-2Z-purinoceptor agonists in human lymphocytes is dependent on bivalent cation influx

被引:65
作者
Gargett, CE
Cornish, EJ
Wiley, JS
机构
[1] AUSTIN & REPATRIAT MED CTR,DEPT HAEMATOL,HEIDELBERG,VIC 3084,AUSTRALIA
[2] DEAKIN UNIV,BURWOOD,VIC 3125,AUSTRALIA
关键词
D O I
10.1042/bj3130529
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The role of bivalent cations in ATP-stimulated phospholipase D (PLD) activity was investigated in human leukaemic lymphocytes. Cells were labelled with [H-3]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 [H-3]phosphatidylbutanol ([H-3]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 [H-3]PBut accumulation. ATP-stimulated [H-3]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 Ba-133(2+) 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+].
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页码:529 / 535
页数:7
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