Phospholipase D activation by P2Z-purinoceptor agonists in human lymphocytes is dependent on bivalent cation influx

Caroline Eve Gargett, E. Jean Cornish, James S. Wiley

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Abstract

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+].

Original languageEnglish
Pages (from-to)529-535
Number of pages7
JournalBiochemical Journal
Volume313
Issue number2
DOIs
Publication statusPublished - 15 Jan 1996
Externally publishedYes

Cite this

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title = "Phospholipase D activation by P2Z-purinoceptor agonists in human lymphocytes is dependent on bivalent cation influx",
abstract = "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+].",
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Phospholipase D activation by P2Z-purinoceptor agonists in human lymphocytes is dependent on bivalent cation influx. / Gargett, Caroline Eve; Cornish, E. Jean; Wiley, James S.

In: Biochemical Journal, Vol. 313, No. 2, 15.01.1996, p. 529-535.

Research output: Contribution to journalArticleResearchpeer-review

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