title: A(2a) adenosine receptor mediates PKA-dependent glutamate release from synaptic-like vesicles and Ca(2+) efflux from an IP(3)- and ryanodine-insensitive intracellular calcium store in astrocytes.
abstract: OBJECTIVE
  The mechanism underlying transmitter release from astrocytes is not fully understood. The present study examined A(2a) adenosine receptor-mediated glutamate release and intracellular Ca(2+) rise in cultured rat hippocampal astrocytes.
  
  
  METHODS
  Intracellular amino acids were measured with HPLC. Glutamate release from astrocytes and intracellular Ca(2+) mobilizations were monitored in the NADH imaging, FM1-43 imaging, and fura-2 imaging. The siRNA to silence the A(2a) adenosine receptor-targeted gene was constructed and transfected into cells.
  
  
  RESULTS
  Glutamate was condensed in 'synaptic-like vesicle' fractions. In the NADH imaging, CGS21680, an agonist of A2a adenosine receptors, increased NADH fluorescent signals, that reflects glutamate release, and the effect was inhibited by DMPX, an inhibitor of A(2a) adenosine receptors, H-89, a PKA inhibitor, vesicular transport inhibitors, or botulinum toxin-A, an exocytosis inhibitor. In the FM1-43 imaging to see vesicular recycling, CGS21680 decreased FM1-43 fluorescent signals, that was also prevented by DMPX, H-89, vesicular transport inhibitors, or botulinum toxin-A. CGS21680 increased intracellular Ca(2+) concentrations both in Ca(2+)-containing and -free extracellular solution. The Ca(2+) rise was inhibited by DMPX, H-89, or the vesicular transport inhibitor brefeldin A, but it was not affected by inhibitors for phospholipase C, IP(3) receptor, and ryanodine receptor. CGS21680-induced glutamate release and intracellular Ca(2+) rise were prevented by knocking-down A(2a) adenosine receptor.
  
  
  CONCLUSIONS
  The results of the present study show that A(2a) adenosine receptor/PKA promotes glutamate release from synaptic-like vesicles and stimulates Ca(2+) efflux from an IP(3)- and ryanodine-insensitive intracellular calcium store.
authors.affiliation: Division of Bioinformation, Department of Physiology, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Kanno
authors.forename: Tomoyuki
authors.initials: T
authors.lastname: Nishizaki
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives / Q000494:pharmacology; D058908:Adenosine A2 Receptor Agonists / Q000494:pharmacology; D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D001253:Astrocytes / Q000378:metabolism*; D020013:Calcium Signaling; D002458:Cell Fractionation; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism*; D006624:Hippocampus / Q000166:cytology; D015544:Inositol 1,4,5-Trisphosphate / Q000502:physiology; D010627:Phenethylamines / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D043705:Receptor, Adenosine A2A / Q000235:genetics / Q000378:metabolism*; D012433:Ryanodine / Q000494:pharmacology; D050600:SNARE Proteins / Q000378:metabolism; D022142:Secretory Vesicles / Q000378:metabolism*; D050598:Vesicular Glutamate Transport Protein 1 / Q000378:metabolism; D050599:Vesicular Glutamate Transport Protein 2 / Q000378:metabolism; D050496:Vesicular Glutamate Transport Proteins / Q000378:metabolism
chemical_list: D058908:Adenosine A2 Receptor Agonists; D000596:Amino Acids; D010627:Phenethylamines; D043705:Receptor, Adenosine A2A; D050600:SNARE Proteins; C493475:Slc17a6 protein, rat; C493466:Slc17a7 protein, rat; C482103:Slc17a8 protein, rat; D050598:Vesicular Glutamate Transport Protein 1; D050599:Vesicular Glutamate Transport Protein 2; D050496:Vesicular Glutamate Transport Proteins; C061282:2-(4-(2-carboxyethyl)phenethylamino)-5'-N-ethylcarboxamidoadenosine; D012433:Ryanodine; D018698:Glutamic Acid; D015544:Inositol 1,4,5-Trisphosphate; D017868:Cyclic AMP-Dependent Protein Kinases; D000241:Adenosine