Activation of the Adenosine A3Receptor in RAW 264.7 Cells Inhibits Lipopolysaccharide-Stimulated Tumor Necrosis Factor-α Release by Reducing Calcium-Dependent Activation of Nuclear Factor-κB and Extracellular Signal-Regulated Kinase 1/2

Bacterial lipopolysaccharide (LPS) activates the immune system and promotes inflammation via Toll-like receptor (TLR) 4, which regulates the synthesis and release of tumor necrosis factor (TNF)-α and other inflammatory cytokines. Previous studies have shown that the nucleoside adenosine suppresses LPS-stimulated TNF-α release in human UB939 macrophages by activating an adenosine A₃ receptor (A₃AR) subtype on these cells. In this study, we examined the mechanism(s) underlying A₃AR-dependent inhibition of TNF-α release in a mouse (RAW 264.7) cell line. Treatment of RAW 264.7 cells with LPS (3 μg/ml) increased TNF-α release, which was reduced in a dose-dependent manner by adenosine analogs N⁶-(3-iodobenzyl)-adenosine-5′-N-methyluronamide (IB-MECA) and R-phenylisopropyladenosine and reversed by selective A₃AR blockade. The increase in TNF-α release was preceded by an increase in intracellular Ca²⁺ levels. Inhibition of intracellular Ca²⁺ release by IB-MECA, a selective agonist of the A₃AR, or with BAPTA-AM, an intracellular Ca²⁺ chelator, reduced LPS-stimulated TNF-α release. Activation of the A₃AR or inhibition of intracellular Ca²⁺ release also reduced LPS-stimulated nuclear factor-κB (NF-κB) activation and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. Similar inhibition by A₃AR was observed for LPS-stimulated inducible nitric-oxide synthase. These data support the contention that inhibition of LPS-stimulated release of inflammatory molecules, such as TNF-α and NO via the A₃AR, involves suppression of intracellular Ca²⁺signaling, leading to suppression of NF-κB and ERK1/2 pathways.