Role of P2X7 receptors in signaling and eATP efflux by chondrocytes. (A) Chondrocytes were treated with no additives (control) or P2X7 inhibitors (Brilliant Blue G (BBG), AZ10606120 or A438079) for 1 h in the presence of 1 mM ATP. Prostaglandin E2 (PGE2) levels in the media were measured using the Parameter™ Prostaglandin E2 kit (R&D Systems). Bars represent mean ± standard error. BBG reduced ATP-induced PGE2 levels (n = 8; ***P <0.001). (B) Chondrocytes were transfected with siRNA for P2X7 or a scrambled control. After 48 h, cells were exposed to hypotonic media for 10 minutes (gray bars) or isotonic media (black bars) and eATP levels were measured. Bars represent mean ± standard error. Under control conditions, a hypotonic challenge consistently increases (eATP). No differences in eATP levels were noted in siP2X7-treated chondrocyte media (n = 8; P >0.05). In parallel cultures at 48 h, protein and mRNA were isolated from scramble or siRNA-treated cells as described and used to assess levels of P2X7 receptor protein (C) and mRNA (D). mRNA levels of P2X7 were suppressed in siRNA-treated cells (*P <0.05). Western blots compare the effects of siRNA effects on P2X7 receptor levels versus actin.