title: Diverse signal transduction pathways mediated by endogenous P2 receptors in cultured rat cerebral cortical neurons.
abstract: The present study was conducted to assess the intracellular signaling pathways mediated by receptors for ATP, uridine triphosphate (UTP), and 2-methylthio ATP (2-MeSATP), by monitoring patch-clamp currents and intracellular calcium mobilization in cultured rat cortical cerebral neurons. All three agonists evoked potassium currents and increased the intracellular free Ca2+ concentration ([Ca2+]i), and these effects were inhibited by the broad G-protein inhibitor guanosine-5'-O-(2-thiodiphosphate) (GDPbetaS) but not by the Gi/o-protein inhibitor pertussis toxin (PTX). UTP-evoked currents were inhibited by either the phospholipase C inhibitor neomycin or the selective protein kinase C (PKC) inhibitor GF109203X, and the rise in cytosolic Ca2+ was inhibited by either neomycin or the inositol 1,4,5-trisphosphate (IP3) receptor antagonist heparin, indicating that the UTP receptor involved phospholipase C-mediated phosphatidylinositol signaling. In contrast, 2-MeSATP-induced currents and rise in cytosolic Ca2+ were not inhibited by either neomycin, or GF109203X, or heparin. 2-MeSATP elicited single-channel currents in the cell-attached patch-clamp configuration and also in excised patches. The G-protein activator GTP gamma S induced single-channel currents in a fashion that mimicked the effect of 2-MeSATP. These data suggest that 2 MeSATP activated potassium channels by a direct action of G-protein beta gamma subunits and increased [Ca2+]i by a mechanism independent of phospholipase C stimulation and IP3 production. ATP-evoked currents were partially inhibited by either neomycin or GF109203X, although the rise in cytosolic Ca2+ was not affected by these inhibitors. ATP produced single-channel currents with two major classes of the slope conductance (86 and 95 pS) in cell-attached patches, each of which is consistent with that achieved by 2-MeSATP (85 pS) or UTP (96 pS); the currents with the lower conductance were observed in the outside-out patch-clamp configuration. These results indicate that P2 receptors for UTP and 2-MeSATP are linked to a PTX-insensitive G-protein involving different signal transduction pathways and that ATP responses are mediated by both of these P2 receptors.
authors.affiliation: Department of Physiology, Kobe University School of Medicine, Chuo-ku, Kobe 650, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Nishizaki
authors.forename: M
authors.initials: M
authors.lastname: Mori
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D000831:Animals, Newborn; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology*; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000187:drug effects; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006493:Heparin / Q000494:pharmacology; D007211:Indoles / Q000494:pharmacology; D017136:Ion Transport / Q000187:drug effects; D008301:Maleimides / Q000494:pharmacology; D009355:Neomycin / Q000494:pharmacology; D009419:Nerve Tissue Proteins / Q000502:physiology; D009474:Neurons / Q000187:drug effects* / Q000502:physiology; D018408:Patch-Clamp Techniques; D037342:Pertussis Toxin; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D011188:Potassium / Q000378:metabolism; D015221:Potassium Channels / Q000187:drug effects / Q000502:physiology; D058910:Purinergic P2 Receptor Agonists; D051381:Rats; D018048:Receptors, Purinergic P2 / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology*; D012964:Sodium / Q000378:metabolism; D013873:Thionucleotides / Q000494:pharmacology; D010738:Type C Phospholipases / Q000378:metabolism; D014544:Uridine Triphosphate / Q000494:pharmacology; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
chemical_list: D007211:Indoles; D008301:Maleimides; D009419:Nerve Tissue Proteins; D015221:Potassium Channels; D058910:Purinergic P2 Receptor Agonists; D018048:Receptors, Purinergic P2; D013873:Thionucleotides; D010566:Virulence Factors, Bordetella; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000255:Adenosine Triphosphate; D006493:Heparin; D012964:Sodium; D037342:Pertussis Toxin; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D009355:Neomycin; C070515:bisindolylmaleimide I; D011188:Potassium; D002118:Calcium; D014544:Uridine Triphosphate; C008501:2-methylthio-ATP