Figure 1
Effect of a hypotonic challenge on eATP levels in media from monolayer and agarose cultures and characterization of putative ATP transport participants. (A) Normal adult porcine chondrocytes were plated in high-density short-term monolayer cultures. Media were replaced with fresh media (control) or identical quantities of media containing increasing percentages (volume/volume) of H2O. eATP levels were measured 10 minutes post-treatment. Bars represent mean ± standard error (A-C). eATP levels were increased by 35 and 50% H20 compared to an undiluted media control (n = 8;***P <0.001). (B) Chondrocytes were embedded in 2% agarose. Media were replaced with fresh media (control) or identical quantities of media containing increasing percentages (volume/volume) of H2O. eATP levels were measured 10 minutes after the media change. eATP levels were increased by 35 and 50% H20 compared to undiluted media control (n = 8;***P <0.001). (C) Chondrocytes were exposed to hypotonic media (gray bars) or isotonic media (black bars) for various lengths of time and eATP levels were measured. eATP levels were increased compared to unchallenged control at 10 minutes and 1 h (n = 8;***P <0.001, **P <0.01). D. Chondrocyte protein extracts were run in western blots using specific antibodies against pannexin 1 and 3, ANK, P2X7 and P2X4. Total RNA was reverse transcribed and analyzed for mRNA for pannexin 1, ANK, P2X7 and P2X4 as described. The final PCR products were electrophoresed on a 1% ethidium bromide-stained agarose gel. Due to absence of a sequence for porcine pannexin-3, RT-PCR for pannexin-3 was not performed.