Induction of the P2z/P2X7 Nucleotide Receptor and Associated Phospholipase D Activity by Lipopolysaccharide and IFN-γ in the Human THP-1 Monocytic Cell Line

Benjamin D. Humphreys, George R. Dubyak

Research output: Contribution to journalArticle

154 Scopus citations

Abstract

The activation of phospholipase D (PLD) was used as a marker for P2z nucleotide receptor (P2zR) activity in the human THP-1 monocytic cell line. While untreated THP-1 cells displayed little PLD response to the P2zR agonist 3′-O-(4-benzoyl)benzoyl (Bz)ATP, treatment with either IFN-γ or bacterial LPS induced a BzATP-mediated stimulation of PLD activity. Treatment of cells with combinations of IFN-γ and LPS resulted in synergistic induction. P2z receptors mediated these effects because: 1) reduction of extracellular divalent cation concentration increased agonist potency; 2) only BzATP or ATP acted as agonist nucleotides; 3) oxidized ATP, an inhibitor of the P2z receptor, abolished the response. The P2zR-stimulated PLD was rapidly activated (t1/2 = 1.5 min), completely inhibited by KN-62, a calcium-calmodulin kinase II inhibitor, and only partially repressed by bisindolylmaleimide, a protein kinase C inhibitor. The P2zR-mediated PLD activity was distinguished from phorbol ester-stimulated PLD activity because the latter was slowly activated (t1/2 > 15 min), unaffected by oxidized ATP or KN-62, and completely inhibited by bisindolylmaleimide. IFN-γ and LPS treatment also synergistically induced P2zR-dependent changes in membrane permeability and cytolysis, as indicated by BzATP-mediated Ca2+ influx, ethidium bromide uptake, and lactate dehydrogenase release. Finally, IFN-γ and LPS synergistically up-regulated mRNA encoding the P2X7 receptor, a recently cloned ATP-gated channel that exhibits a P2zR phenotype.

Original languageEnglish
Pages (from-to)5627-5637
Number of pages11
JournalJournal of Immunology
Volume157
Issue number12
StatePublished - Dec 15 1996
Externally publishedYes

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