Fig. 7.

TRPV1 induced CDI on Orai1 modulates wound healing. a Cell population calcium measurements obtained with cortical astrocytes primary cultures transfected with Orai1-GCaMP3. Endogenous TRPV1 and P2X₄ were used. Orai1-GCaMP3 senses calcium increments evoked with capsaicin (500 nM, blue) and TG (1 μM, cyan) but not with CTP (black). CTP induces robust calcium increments as reported by R-GECO (magenta). Data shows the mean ± standard deviation from at least 10 independent measurements obtained from 5 different cultures. b Stimulation with capsaicin (500 nM) prior to activation of Orai1 with thrombin induces strong CDI. CTP stimulation induces large P2X₄ currents, however not CDI in Orai1 (activated by thrombin). c Perforated patch measurements of currents evoked by a voltage step from 0 to −100 mV in response to capsaicin followed by thrombin (black lines), thrombin alone (magenta) and capsaicin + thrombin in cells incubated with BAPTA-AM (cyan). BAPTA incubation strongly reduces CDI in cells stimulated with capsaicin and thrombin. Capsaicin incubated for 3 min and then the cells were washed with fresh PBS for 3 min prior to thrombin stimulation. d Perforated patch measurements of currents evoked by a voltage step from 0 to −100 mV in response to capsaicin + thrombin in cells expressing TRPV1 (black) and cells expressing also the ANK1-GFP domain (cyan) and cells incubated with BAPTA-AM (magenta). e Scratch-wound assay in cortical astrocytes. Representative photographs of the cell culture around the wound area are shown for 10 and 25 h after wound. Medium shows the cells that were maintained in DMEM. Thrombin indicates cells treated with thrombin (5 U mL⁻¹ thrombin) for 3 min⁴². Cell cultures were exposed to capsaicin (500 nM) for 3 min and then incubated with thrombin (5 U mL⁻¹ thrombin) for 3 min. f Percentage of confluency in wound area under the different treatments shown in e. Cells were also transfected with a construct carrying ANK1-GFP (blue). Data points show the mean ± standard deviation from at least 5 cell culture dishes for each condition. *p < 0.01. The statistical significance was p < 0.05 according to ANOVA