ADENOSINE A_2A RECEPTOR INHIBITS TNF-a PRODUCTION INDUCED BY Tat BY BLOCKING CALCIUM RELEASE FROM IP₃ RECEPTORS VIA PROTEIN PHOSPHATASE

M Mayne*^1,2, J Fotheringham^1,2, C Holden¹, A Nath³, JD Geiger^1,2
1Department of Pharmacology and Therapeutics, University of Manitoba, Winnipeg, Manitoba; Division of Neurovirology and Neurodegenerative Disorders, St Boniface Hospital Research Centre, Winnipeg, Manitoba; ³Department of Neurology, ²Microbiology and Immunology, Lexington, Kentucky

Background: Because HIV Tat is associated with increased neuroinflammation and neurotoxicity, there is a keen interest in determining the cellular events regulated by Tat.
Objectives: Because we have shown previously that Tat-induced TNF-a production is dependent on intracellular calcium released from the inositol trisphosphate (IP₃) pool and that adenosine is a potent inhibitor of TNF-a, we determined the extent to which adenosine receptors regulate calcium release from endoplasmic reticulum pools.
Methods: Primary human macrophages were purified, differentiated for 7 d. Cells were treated with adenosine agonists for 30 min and Tat-induced intracellular calcium release was measured by Fura-2 imaging and TNF-a production by ELISA.
Results: Activation of the A_2A receptor using CGS 21680 (100 nM) inhibited Tat-induced elevation of intracellular calcium by 90±8%. The actions of CGS 21680 treatment were not reversed by the adenylyl cyclase inhibitor SQ22536 or the protein kinase A specific inhibitor H89. A_2A receptor-regulated inhibition of calcium release from IP₃R-regulated stores was dependent on protein phosphatase activity as okadaic acid (1 mM) reversed the actions of CGS21680. Finally, CGS21680 potently inhibited Tat-induced TNF-a production in primary macrophages by 90±6% and importantly, okadaic acid reversed the inhibitory actions of A_2A.
Conclusions: These data indicate that A_2A receptor activation blocks the release of calcium from IP₃R-regulated stores via activation of protein phosphatase and that the potent anti-inflammatory actions of A_2A including inhibition of TNF-a production are mediated primarily through protein phosphatase activation.