Figure 2.

Potential participation of Ca²⁺ and Na⁺ ions in HGH solution-induced HPMC death. HPMC were exposed to a control (300 mOsM) condition or exposed to 345, 395, or 485 mOsM HGH solutions for 24 h. Then, both intracellular Ca²⁺ (A,B) and Na⁺ (C) levels were determined by means of the Ca²⁺ sensitive dye Fluo-3 (5 μM) (A) and Fura-Red (15 μM) (B) and the Na⁺-sensitive dye CoroNa Green-AM (5 μM) (C). Statistical differences were assessed by one-way ANOVA (or the non-parametric Kruskal–Wallis method) followed by Dunn's post-hoc test. ^*P < 0.05 and ^**P < 0.01 against control condition (300 mOsM). Graph bars show the mean ± SD (N = 5 independent cultures). HPMC were cultured in the absence (control solution, open bars) or presence of 10 μM 2-APB (gray bars, D,E), 1 μM nidefipine (filled bars, D,E), 1 μM ambroxol (gray bars, F), and 1 μM amiloride (filled bars, F), and then exposed to the control (300 mOsM) condition or to the 485 mOsM HGH solution for 24 h. Then, both intracellular Ca²⁺ (D,E) and Na⁺ (F) levels were determined by means of the Ca²⁺ sensitive dye 5 μM Fluo-3 (D) and 15 μM Fura-Red (E) and the Na⁺-sensitive dye 5 μM CoroNa Green-AM (F). Statistical differences were assessed by two-way ANOVA followed by Tukey's post-hoc test. ^*P < 0.05 against control condition (300 mOsM). Graph bars show the mean ± SD (N = 7 independent cultures). HPMC death was determined in cells cultured in the absence (unfilled bars) or presence (filled bars) of a Ca²⁺-free solution (4 h pulse) (G), BAPTA-AM solution (5 μM, 4 h pulse) (H), and a Na⁺-free solution (I), and exposed to the control (300 mOsM) condition or the 485 mOsM HGH solution for 24 h. Statistical differences were assessed by two-way ANOVA followed by Tukey's post-hoc test. ^**P < 0.01 against control condition (300 mOsM). Graph bars show the mean ± SD (N = 7 independent cultures). Cell membrane depolarization was measured by the indicator DiBAC₄(3) in cells exposed to the control (300 mOsM) condition or the 485 mOsM HGH solution (J). Statistical differences were assessed by the student's t-test (Mann-Whitney). ^**P < 0.01 against control condition (300 mOsM). Graph bars show the mean ± SD (N = 8 independent cultures). HPMC death was determined in cells cultured in the absence (control solution, unfilled bars) or presence of 10 μM 2-APB (gray bars, K), 1 μM nidefipine (filled bars, K), 1 μM ambroxol (gray bars, L), and 1 μM amiloride (filled bars, L), and then exposed to the control (300 mOsM) condition or the 485 mOsM HGH solution for 24 h. Statistical differences were assessed by two-way ANOVA followed by Tukey's post-hoc test. ^*P < 0.05 against control condition (300 mOsM). Graph bars show the mean ± SD (N = 6 independent cultures). All reagents were added 1 h before HGH solution exposure and were maintained during the experiments.