Journal
CELLULAR PHYSIOLOGY AND BIOCHEMISTRY
Volume 30, Issue 6, Pages 1398-1412Publisher
KARGER
DOI: 10.1159/000343328
Keywords
A(2a) adenosine receptor; Protein kinase A; Glutamate release; Synaptic-like vesicles; Intracellular Ca2+ rise
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Background/Aims: The mechanism underlying transmitter release from astrocytes is not fully understood. The present study examined A(2a) adenosine receptor-mediated glutamate release and intracellular Ca2+ rise in cultured rat hippocampal astrocytes. Methods: Intracellular amino acids were measured with HPLC. Glutamate release from astrocytes and intracellular Ca2+ 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 A(2a) 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 Ca2+ concentrations both in Ca2+-containing and -free extracellular solution. The Ca2+ rise was inhibited by DMPX, H-89, or the vesicular transport inhibitor brefeldin A, but it was not affected by inhibitors for phospholipase C, IP3 receptor, and ryanodine receptor. CGS21680-induced glutamate release and intracellular Ca2+ rise were prevented by knocking-down A(2a) adenosine receptor. Conclusion: The results of the present study show that A(2a) adenosine receptor/PKA promotes glutamate release from synaptic-like vesicles and stimulates Ca2+ efflux from an IP3- and ryanodine-insensitive intracellular calcium store. Copyright (c) 2012 S. Karger AG, Basel
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