title: Translocation of neuronal nitric oxide synthase to the plasma membrane by ATP is mediated by P2X and P2Y receptors.
abstract: BACKGROUND
  The translocation of neuronal nitric oxide synthase (nNOS) from the cytosol to the membrane is functionally coupled to the activation of N-methyl-D-aspartate (NMDA) receptors at synapses. Whereas there is abundant evidence indicating that ATP and nitric oxide are involved in nociceptive transmission, whether nNOS is activated by ATP remains unknown. We recently established a fluorescence imaging system for examining nNOS translocation in PC12 cells expressing a yellow fluorescence protein-tagged nNOS N-terminal mutant, nNOSNT-YFP, and examined the effect of ATP on nNOS translocation using the system.
  
  
  RESULTS
  The translocation of nNOS was induced by ATP in the presence of NMDA and forskolin, an adenylate cyclase activator. The purinergic P2X receptor agonist 2-MeSATP and the P2Y agonist UTP significantly enhanced nNOS translocation; and simultaneous stimulation with 2-MeSATP and UTP exhibited the same concentration-response curve for the translocation as obtained with ATP. ATP, 2-MeSATP, and UTP increased the intracellular Ca2+ concentration ([Ca2+]i) in PC12 cells. Conversely, whereas the P2X receptor antagonist PPADS and the P2Y antagonist reactive blue-2 partially inhibited increases in the translocation of nNOS and [Ca2+]i by ATP, the non-selective P2 receptor antagonist suramin completely blocked them. In addition, the increase in the nNOS translocation by ATP was blocked by NMDA receptor antagonists and inhibitors of protein kinase A, protein kinase C, and Src kinase. Consistent with the expression of P2X and P2Y receptors in the spinal cord, ATP and UTP increased the [Ca2+]i in primary cultured spinal neurons. ATP potentiated and prolonged the [Ca2+]i increase produced by NMDA in the dorsal horn of the spinal cord. Furthermore, the selective P2X3/P2X2/3 antagonist A-317491 inhibited nNOS activation assessed by NO formation in spinal slices prepared from neuropathic pain model mice.
  
  
  CONCLUSIONS
  ATP is involved in nNOS translocation mediated by protein kinase C via activation of P2X and P2Y receptors and nNOS translocation may be an action mechanism of ATP in nocieptive processing in the spinal cord.
authors.affiliation: Department of Medical Chemistry, Kansai Medical University, Fumizono, Moriguchi, Japan. ohnishit@med.niigata-u.ac.jp
authors.forename: Takayuki
authors.initials: T
authors.lastname: Ohnishi
authors.forename: Shinji
authors.initials: S
authors.lastname: Matsumura
authors.forename: Seiji
authors.initials: S
authors.lastname: Ito
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D002462:Cell Membrane / Q000201:enzymology* / Q000378:metabolism; D051379:Mice; D052248:Nitric Oxide Synthase Type I / Q000378:metabolism*; D010146:Pain / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D021381:Protein Transport; D018048:Receptors, Purinergic P2 / Q000378:metabolism*; D058469:Receptors, Purinergic P2X; D058472:Receptors, Purinergic P2Y1; D013116:Spinal Cord
chemical_list: C546383:P2ry1 protein, mouse; D018048:Receptors, Purinergic P2; D058469:Receptors, Purinergic P2X; D058472:Receptors, Purinergic P2Y1; D000255:Adenosine Triphosphate; D052248:Nitric Oxide Synthase Type I; C496321:Nos1 protein, mouse; D011493:Protein Kinase C