Figure 2.

Industrial isocyanates and tear gas agents activate native TRPA1 channels in cultured sensory neurons. A) Industrial isocyanates induced Ca²⁺ influx into cultured mouse DRG neurons, as measured by fluorescent Fura-2 imaging. Neurons are shown before activation (Pre, left column), 70 s after challenge (middle column) with MIC (100 μM, top row) or HDI (100 μM, bottom row), and after application of 5 μM capsaicin (Cap, right column) after 50 s. Pseudocolors denote 0–3 μM [Ca²⁺]_i. Original view, ×10. B) Average [Ca²⁺]_i of mouse DRG neurons (thick lines) with an application of MIC (100 μM, n=168 neurons from 2 mice, black line) or HDI (100 μM, n=270 from 2 mice, red line), followed by 100 μM mustard oil, 5 μM capsaicin (Cap), and 65 mM KCl. Thin lines represent se. C) Tear gas agent-induced Ca²⁺ influx into cultured murine DRG neurons, as measured by fluorescent Fura-2 imaging. Neurons are shown before activation (Pre, left column), 70 s after challenge (middle column) with CS (100 μM, top row) or CN (100 μM, middle row) or CR (300 μM, bottom row) and after application of 5 μM capsaicin (Cap, right column) after 50 s. Pseudocolors denote 0–3 μM [Ca²⁺]_i. Original view, ×10. D) Average [Ca²⁺]_i of mouse DRG neurons (thick lines) with an application of CS (100 μM, blue line, n=161 neurons from 2 mice), CN (100 μM, green line, n=335 from 5 mice), or CR (300 μM, red line, n=137 from 2 mice), followed by 100 μM mustard oil, 5 μM capsaicin (Cap), and 65 mM KCl. Thin lines represent se. E) Dose-response curves of isocyanate-activated Ca²⁺-influx into mouse DRG neurons are similar to hTRPA1-transfected HEK-293T cells. [Ca²⁺]_i induced by each dose is represented as percentage of maximal [Ca²⁺]_i elicited by a saturating dose of mustard oil (100 μM) applied 75 s later (baseline [Ca²⁺]_i was subtracted). Mustard oil-sensitive mouse DRG neurons were activated by MIC (EC₅₀=36±7 μM, n=30±6 neurons/dose, solid black squares) and HDI (EC₅₀=8.4±1.4 μM, n=39±12 /dose, solid red squares). Dashed lines and open circles represent hTRPA1-transfected HEK-293T cells, as shown in Fig. 1C. F) Dose-response curves of tear gas agent-activated Ca²⁺-influx into mouse DRG neurons are right-shifted compared with responses in hTRPA1-transfected HEK293T cells. [Ca²⁺]_i represented as in E. Mustard oil-sensitive mouse DRG neurons were activated by CS (EC₅₀=12.1±0.3 μM, n=41±9 neurons/dose, solid blue squares), CN (EC₅₀ =6±1 μM, n=23±5 /dose, solid green squares), and CR (EC₅₀= 246±27 μM, n=37±16 /dose, solid red squares). Dashed lines with open circles represent dose-response curves of Ca²⁺-influx into hTRPA1-transfected HEK-293T cells shown in Fig. 1D.