Fig. 4

The TRPA1 ion channel is functional in primary human OA chondrocytes (a, b) and human T/C28a2 chondrocyte cell line (c) as shown by TRPA1-mediated Ca²⁺ influx. Primary human chondrocytes (a, b) and human T/C28a2 chondrocytes (c) were cultured with or without (control) IL-1β (100 pg/ml) for 24 h. HEK293 cells transfected with plasmids encoding human TRPA1 were used as positive control cells (d). The cells were loaded with Fluo-3-AM and the TRPA1-mediated Ca²⁺ influx was measured by Victor3 multilabel counter at excitation/emission wavelengths of 485/535 nm at 1/s frequency. The cells were first preincubated with the TRPA1 antagonist HC-030031 (100 μM) or the vehicle for 30 min at +37 °C. In the measurements, basal fluorescence was first recorded for 15 s and thereafter the selective TRPA1 agonist allyl isothiocyanate (AITC; 50 μM) was added and the measurement was continued for 30 s after which the control ionophore compound ionomycin (1 μM) was introduced to the cells. IL-1β stimulation resulted in an elevation in AITC-induced Ca²⁺ influx compared to unstimulated control cells, and it was attenuated by the selective TRPA1 antagonist HC-030031. The results were normalized against the background and expressed as mean of eight simultaneous measurements. Curves in A, C and D express results from one representative experiment. In (b) area under the curve (AUC) from 15 to 45 s was calculated from measurements of primary chondrocyte from four donors (each with eight repeats). Results are expressed as mean + SEM. Repeated measures ANOVA followed by Dunnett’s post-test was used in the statistical analysis; ^** p < 0.01 compared to the IL-1β-treated samples. IL interleukin, OA osteoarthritis, TRPA1 transient receptor potential ankyrin 1