title: Contribution of P2X7 receptors to adenosine uptake by cultured mouse astrocytes.
abstract: Nucleotides and nucleosides play important roles by maintaining brain homeostasis, and their extracellular concentrations are mainly regulated by ectonucleotidases and nucleoside transporters expressed by astrocytes. Extracellularly applied NAD(+) prevents astrocyte death caused by excessive activation of poly(ADP-ribose) polymerase-1, of which the molecular mechanism has not been fully elucidated. Recently, exogenous NAD(+) was reported to enter astrocytes via the P2X7 receptor (P2X7R)-associated channel/pore. In this study, we examined whether the intact form of NAD(+) is incorporated into astrocytes. A large portion of extracellularly added NAD(+) was degraded into metabolites such as AMP and adenosine in the extracellular space. The uptake of adenine ring-labeled [(14)C]NAD(+), but not nicotinamide moiety-labeled [(3)H]NAD(+), showed time- and temperature-dependency, and was significantly enhanced on addition of apyrase, and was reduced by 8-Br-cADPR and ARL67156, inhibitors of CD38 and ectoapyrase, respectively, and P2X7R knockdown, suggesting that the detected uptake of [(14)C]NAD(+) resulted from [(14)C]adenosine acting as a metabolite of [(14)C]NAD(+). Pharmacological and genetic inhibition of P2X7R with brilliant blue G, KN-62, oxATP, and siRNA transfection resulted in a decrease of [(3)H]adenosine uptake, and the uptake was also reduced by low concentration of carbenoxolone and pannexin1 selective peptide blocker (10)panx. Taken together, these results indicate that exogenous NAD(+) is degraded by ectonucleotidases and that adenosine, as its metabolite, is taken up into astrocytes via the P2X7R-associated channel/pore.
authors.affiliation: Department of Environmental Biochemistry, Kyoto Pharmaceutical University, 5 Nakauchi-cho, Misasagi, Yamashina-ku, Kyoto, Japan.
authors.forename: Hiroto
authors.initials: H
authors.lastname: Okuda
authors.forename: Youichirou
authors.initials: Y
authors.lastname: Higashi
authors.forename: Kentaro
authors.initials: K
authors.lastname: Nishida
authors.forename: Sadaki
authors.initials: S
authors.lastname: Fujimoto
authors.forename: Kazuki
authors.initials: K
authors.lastname: Nagasawa
mesh_terms: D000241:Adenosine / Q000378:metabolism*; D000818:Animals; D001253:Astrocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D002478:Cells, Cultured; D017630:Connexins / Q000037:antagonists & inhibitors / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005110:Extracellular Space / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D009243:NAD / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D033703:Nucleoside Transport Proteins / Q000187:drug effects / Q000378:metabolism; D011755:Pyrophosphatases / Q000378:metabolism; D034622:RNA Interference / Q000502:physiology; D058486:Receptors, Purinergic P2X7 / Q000235:genetics / Q000502:physiology*
chemical_list: D017630:Connexins; D004791:Enzyme Inhibitors; D009419:Nerve Tissue Proteins; D033703:Nucleoside Transport Proteins; C503631:Panx1 protein, mouse; D058486:Receptors, Purinergic P2X7; D009243:NAD; D011755:Pyrophosphatases; C092064:ectonucleotide pyrophosphohydrolase; D000241:Adenosine