Roles of Ca2+signaling and transient receptor potential vanilloid 4 (TRPV4) channels in hypotonically-induced release of eATP from chondrocytes. For the following experiments, chondrocytes were incubated with each additive for 30 minutes in 100 μl of media with no additives acting as a control. A hypotonic challenge was initiated by removing 35 μl of media, replacing it with 35 μl of H2O. eATP levels were measured after 10 minutes. Bars represent mean ± standard error. Under control conditions, a hypotonic challenge consistently increases (eATP). (A) Chondrocytes were incubated with 100 μM A23187, which increased eATP levels (n = 8; **P <0.01, ***P <0.001). (B) Chondrocytes were incubated with 10 μM bis-N,N,N',N'-tetraacetic acid-AM (BAPTA-AM), which decreased eATP levels after hypotonic challenge compared to control without BAPTA-AM (n = 8; ***P <0.001). (C) Chondrocytes were incubated with 100 nM GSK1016790A, which increased eATP levels in the basal state and after a hypotonic challenge compared to a control without GSK 1016790 (n = 8; ***P <0.001). (D) Chondrocyte protein extracts were run in western blots with TRPV4 antibody (leftpanel). mRNA for TRPV4 was detected with RT-PCR using a specific primer sequence and electrophoresed on a 1% ethidium bromide-stained agarose gel ( right panel). (E) Chondrocytes were incubated with control media (black bars) or media containing an additional 100 mM NaCl (gray bars) for 10 minutes after which eATP levels were measured. A hypertonic challenge did not increase eATP levels compared to the basal state (n = 8; P >0.05).