Role of P2X4 receptors in signaling and eATP release by chondrocytes. (A) Chondrocytes were treated with no additives (control) or 10 μM ivermectin in 100 μl of media for 30 minutes: 35 μl of media were removed and replaced with 35 μl of media (black bars) or 35 μl of H2O (gray bars). eATP levels were measured after 10 minutes. Bars represent mean ± standard error. Under control conditions, a hypotonic challenge consistently increases (eATP). Ivermectin increased eATP levels after a hypotonic challenge (n = 8: ***P <0.001). (B) Chondrocytes were transfected with siRNA for P2X4 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. eATP levels in P2X4 -silenced chondrocyte media were similar to those in the scramble control 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 P2X4 receptor protein (C) and mRNA (D). siRNA for P2X4 decreased P2X4 mRNA (**P <0.01). Western blots compare effects on P2X4 receptor versus actin.