title: A novel pathway for the activation of phospholipase D by P2z purinergic receptors in BAC1.2F5 macrophages.
abstract: Macrophages express two distinct types of nucleotide (P2 purinergic) receptors for extracellular ATP: one type induces a Ca(2+)-mobilizing response via the activation of phosphatidylinositol-phospholipase C (PI-PLC) while the second type induces the rapid formation of nonselective pores which are permeated by ions and small (< 1 kDa) organic molecules. We have confirmed the presence of these two ATP receptor types in the BAC1.2F5 murine macrophage cell line and have identified 3'-O-(4-benzoyl)benzoyl-ATP (BzATP) as a selective and potent agonist for the so-called P2z or pore-forming ATP receptor type. Several lines of evidence indicated that occupation of these P2z receptors is also accompanied by a rapid and large increase in the activity of a phosphatidylcholine-selective phospholipase D (PLD) effector enzyme. In cells metabolically labeled with [3H]oleic acid or [3H]glycerol and stimulated in the presence of ethanol, ATP and BzATP induced a severalfold increase in the rate and extent of [3H]phosphatidylethanol (PEt) accumulation. These responses were stimulated only by ATP, BzATP, and ATP gamma S (adenosine 5'-O-(3-thiotriphosphate) with the rank order of potency: BzATP >> ATP > ATP gamma A; there was no response to other adenine nucleotides or to non-adenine nucleotides. Significantly, the ability of P2z receptor agonists to stimulate this PLD activity was not dependent on the presence of extracellular [Ca2+] or elevation of cytosolic [Ca2+]. The inability of ionomycin, gramicidin, digitonin, UTP, platelet-activating factor, or phorbol ester to quantitatively mimic these nucleotide effects suggested that activation of this PLD by P2z receptor agonists was not a secondary response due to: 1) enhanced Ca2+ influx; 2) membrane depolarization; 3) nonselective permeabilization of the plasma membrane; 4) stimulation of Ca(2+)-mobilizing ATP receptors; 5) stimulation of a primary PI-PLC pathway; or 6) activation of protein kinase C. These findings suggest that activation of a novel PLD-based signaling pathway may play an important role in the modulation of macrophage function by pore-forming P2z receptors for extracellular ATP.
authors.affiliation: Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106.
authors.forename: C
authors.initials: C
authors.lastname: el-Moatassim
authors.forename: G R
authors.initials: GR
authors.lastname: Dubyak
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology*; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002461:Cell Line, Transformed; D002794:Choline / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004072:Digitonin / Q000494:pharmacology; D004075:Diglycerides / Q000378:metabolism; D004789:Enzyme Activation; D007295:Inositol Phosphates / Q000378:metabolism; D015759:Ionomycin / Q000494:pharmacology; D007700:Kinetics; D008264:Macrophages / Q000378:metabolism*; D051379:Mice; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D010739:Phospholipase D / Q000378:metabolism*; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D010972:Platelet Activating Factor / Q000494:pharmacology; D011983:Receptors, Purinergic / Q000187:drug effects / Q000502:physiology*; D013539:Simian virus 40 / Q000235:genetics; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D004075:Diglycerides; D007295:Inositol Phosphates; D010972:Platelet Activating Factor; D011983:Receptors, Purinergic; C033901:3'-O-(4-benzoyl)benzoyladenosine 5'-triphosphate; D015759:Ionomycin; D000255:Adenosine Triphosphate; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; D010739:Phospholipase D; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D004072:Digitonin; D002794:Choline; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium