Figure 1.

ET-1 sensitizes TRPA1 channel exogenously expressed in HEK293 cells. (a) Pseudo color images from Fura-2 ratiometric imaging showing Ca²⁺ responses in HEK293 in response to TRPA1 agonist MO (5 µM) with or without pretreatment of ET-1 (100 nM). Ionomycin (1 µM) was applied at the end of the recording to determine all active cells. HEK293 cells were cotransfected with hTRPA1 and ET_AR. (b) Averaged Ca²⁺ responses from experiments shown in panel (a). Red and black lines depict conditions with or without ET-1 pretreatment, respectively. n > 30 cells/group. (c) Averaged Ca²⁺ responses of HEK293 cells expressing hTRPA1 with ET_AR or with empty vector (pcDNA3.1). n > 30 cells/group. (d) Pharmacological analysis of ET-1-induced TRPA1 sensitization in HEK293 cells. Cells were preincubated with ET_AR antagonist BQ-123 (10 µM), ET_BR antagonist BQ-788 (5 µM), PKA activator forskolin (15 µM), PKA inhibitor H89 (10 µM), PLC inhibitor edelfosine (10 µM), PKC inhibitor BIM (100 nM), or corresponding vehicle (0.1% DMSO) for 5 min, and then these reagents were coapplied with ET-1 (100 nM) during the imaging test. Ca²⁺ responses were normalized to ionomycin applied at the end of the tests for comparison (% response of ionomycin). n = 3–5 tests/group. Each test contains up to 30 cells. ^##p < 0.01 versus control group. **p < 0.01 versus ET-1+Veh group (p > 0.05).

ET-1: endothelin-1; ET_AR: ET_A receptor; hTRPA1: human TRPA1; MO: mustard oil; NS: no significance; Iono: ionomycin; BIM: bisindolylmaleimide.