title: Induction of the P2z/P2X7 nucleotide receptor and associated phospholipase D activity by lipopolysaccharide and IFN-gamma in the human THP-1 monocytic cell line.
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-gamma or bacterial LPS induced a BzATP-mediated stimulation of PLD activity. Treatment of cells with combinations of IFN-gamma 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 (t(1/2) = 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 (t(1/2) > 15 min), unaffected by oxidized ATP or KN-62, and completely inhibited by bisindolylmaleimide. IFN-gamma 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-gamma and LPS synergistically up-regulated mRNA encoding the P2X7 receptor, a recently cloned ATP-gated channel that exhibits a P2zR phenotype.
authors.affiliation: Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University, Cleveland, OH 44106, USA.
authors.forename: B D
authors.initials: BD
authors.lastname: Humphreys
authors.forename: G R
authors.initials: GR
authors.lastname: Dubyak
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D002118:Calcium / Q000502:physiology; D002413:Cations, Divalent; D002460:Cell Line; D004357:Drug Synergism; D005110:Extracellular Space / Q000502:physiology; D015870:Gene Expression; D006801:Humans; D007371:Interferon-gamma / Q000502:physiology*; D015640:Ion Channel Gating / Q000187:drug effects; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008264:Macrophages / Q000502:physiology*; D010739:Phospholipase D / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D018048:Receptors, Purinergic P2 / Q000502:physiology*; D058486:Receptors, Purinergic P2X7; D015398:Signal Transduction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D002413:Cations, Divalent; D008070:Lipopolysaccharides; C546468:P2RX7 protein, human; D012333:RNA, Messenger; D018048:Receptors, Purinergic P2; D058486:Receptors, Purinergic P2X7; C033901:3'-O-(4-benzoyl)benzoyladenosine 5'-triphosphate; D007371:Interferon-gamma; D000255:Adenosine Triphosphate; D010739:Phospholipase D; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium