Figure 5

ROS Activation Increases TRPV1. A - H ₂ O ₂ Oxidative Burst Is Blocked by PBN. An oxidative burst in SW982 synoviocytes was activated by the application of H₂O₂ (1 mM, arrow) to the media (upper trace). The generated ROS were detected with live cell imaging of the oxidative conversion of the ROS sensitive CM-H2DCFDA fluorophore to DCF over time (min). Presence of the ROS scavenger PBN (2 mM for 2 h, middle trace) in the media prevented the oxidative burst and detected ROS levels were similar to the spontaneous levels seen with the addition of vehicle (with PBN, lower trace). The small error bars are obscured by the large symbols. Numbers of cells are indicated in parenthesis in the legend imaged in three experiments. B - ROS Induced Increase in TRPV1. Immunostaining for TRPV1 in SW982 synoviocytes was significantly increased (arbitrary units) compared to control cultures within 30 min after treatment with the ROS donor, tBOOH. The TRPV1 antagonist, SB-366791, inhibits the staining increase. The effect of the tBOOH when combined with the SB-366791 (30 μM) was not significantly different from the controls. All experiments were done in triplicate. *p < 0.01 compared to the control cultures. C - The TRPV1 expression increase was measureable by Western blot analysis at 8 h. The protein level (normalized with β-actin) in SW982 synoviocytes was increased almost 2-fold after pulsing with 1 mM H₂O₂ for 10 min. All experiments were done in triplicate.