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

摘要

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 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 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. Conclusion: 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.