FIGURE 4.

Activation of TRPV4 causes ATP release from cultured astrocytes. A, schematics representing the biosensor system. HEK293 cells expressing P2X₂ (the biosensor) are placed in close proximity to the astrocytes, then 4αPDD (10 μm) is applied to the astrocytes. At the end of each experiment, the reliability of the biosensors is confirmed by directly applying ATP to a location that is distant to the astrocytes. B, representative traces showing that application of 4αPDD evokes whole-cell current responses (left panel) with inward rectification (red traces labeled b in the right panel) in HEK293 cells transfected with a P2X₂ cDNA. Application of ATP (100 μm) was used to confirm P2X₂-mediated responses (left panel) with inward rectification (green traces labeled c in the right panel). Blue traces labeled a in the right panel represent control currents. Ramp pulses (from −80 mV to +80 mV for 500 ms) were applied at 5-s intervals. The holding potential was −60 mV. The traces are representative of a typical experiment (6 of 43 trials). C, real-time ATP imaging was performed in cultured astrocytes (almost confluent). We stacked all of the images after the experiments were completed. The ratio ATP release is represented as pseudo color images. In this figure the height and color (as shown by the scale bars) represent the amount of ATP that is released. Representative images show ATP release after application of 4αPDD (right panel) and in basal conditions (left panel). D, quantification of ATP release in three representative cultures of WT (left panel) and TRPV4KO (right panel) astrocytes after application of 4αPDD.