Fig. 4

Reduction in activation-induced desensitization of TRPM8 is mediated by MOR and PKC. a Representative calcium imaging traces to two repeated applications of cold (20 °C, blue) or menthol (100 μM, green) in HEK cells transfected with TRPM8 + MOR, and exposed to morphine (MS, 0.5 μM) overnight. b Whisker plots showing the mean ratio (R2/R1) of the amplitude response to cold (66.97 ± 1.37% in control, light blue, vs 93.49 ± 0.95% in MS, dark blue; n = 85 and 103, respectively) or menthol (58.13 ± 1.66% in control, light green vs 96.58 ± 0.94%, in MS, dark green, and 59.4 ± 1.74%, half symbols, with MS + naloxone; n = 93, 111 and 71, respectively in HEK cells transfected with MOR+TRPM8 or in the absence of MOR (60.2 ± 2.17% (n = 66) and 61.58 ± 1.52% (n = 60) in control conditions and after MS overnight, respectively). c Whisker plots showing the mean ratio (R2/R1) of the amplitude response to menthol in transfected HEK cells exposed to the different conditions of treatment: control (59.93 ± 1.41%, n = 100); MS (94.45 ± 0.98%, n = 185), MS + PLC inhibitor U73122 (1 μM; 64.12 ± 1.66%, n = 82), PKC activator PMA (1uM; 94.9 ± 1.04, n = 85), MS + PKC blocker GF109203X (3 μM; 62.62 ± 1.7%, n = 66), MS+ PKCβ blocker enzastaurin (1 μM; 54.58 ± 1.69%, n = 85); MS + ROCK inhibitor Y27632 (1 μM; 105.27 ± 2.01%, n = 55) and MS + PKA inhibitor H89 (100 nM; 97.46 ± 1.9%, n = 92). Error bars indicate ±SEM. Statistical analysis was performed using the Kruskal-Wallis ANOVA followed by the Dunn-Bonferroni post hoc test (**p < 0.05)