Fig 2: Effects of cigarette smoke extract (CSE) treatment on the mRNA and protein expression of Na+/H+ exchanger-1 (NHE-1) in esophageal cell lines. Metaplastic (CP-A) and dysplastic (CP-D) esophageal cell lines were treated with dif-ferent concentrations of CSE (1 and 10 µg/mL) for 6, 24 and 72 h, and the relative gene (a) and protein (b) expressions of NHE-1 were investigated by real-time PCR and Western blot, respectively. GAPDH was used as a protein-loading control. Data represent mean ± SEM of three independent experiments; a = p = 0.05 vs. control.

Fig 3: Effects of cigarette smoke extract (CSE) treatment on the activity of Na+/H+ exchanger-1 (NHE-1) in esophageal cell lines. Metaplastic (CP-A) and dysplastic (CP-D) esophageal cell lines were pretreated with different concentrations of CSE (1, 10 and 100 µg/mL) for 1 h, and the activity of NHE-1 was measured. (a) Representative intracellular pH (pHi) curves present the recovery from acidosis in CP-A cells. (b) Summary data of the calculated activity of NHE-1 in the different cell lines. The rate of pH recovery (J(B-)) was calculated as described in Figure 2b. Data are presented as the mean ± SEM; a = p = 0.05 vs. control; n = 12–14 exp./66–68 ROIs.

Fig 4: Effects of cigarette smoke extract (CSE) treatment and smoking on the activity of Na+/H+ exchanger-1 (NHE-1) in guinea pig esophageal epithelial cells (EECs). (a) Normal guinea pig EECs were pretreated with different concentrations of CSE (1, 10 and 100 µg/mL) for 1 h and the activity of NHE-1 was measured. Representative intracellular pH (pHi) curves show the recovery from acidosis. (b) Summary data of the calculated activity of NHE-1 in guinea pig EECs. The rate of pH recovery (J(B-)) was calculated as described in Figure 2b. Data are presented as the mean ± SEM; a: p = 0.05 vs. control; n = 13–18 exp./86–99 ROIs. (c) Chronic effect of cigarette smoking was investigated, using smoking chamber. Guinea pigs were exposed to whole body cigarette smoke 4 times a day, 5 days a week, 30 min each time, using a TE2 closed-chamber manual smoking system. After 1, 2, or 4 months of smoking, the animals were sacrificed and NHE-1 activity was measured. (d) Summary data of the calculated activity of NHE-1 in guinea pig EECs. The rate of pH recovery (J(B-)) was calculated as described in Figure 2b. Data are presented as the mean ± SEM; a: p = 0.05 vs. control; n = 8–16exp./81–210 ROIs.

Fig 5: Expression of Na+/H+ exchanger-1 (NHE-1) in human esophageal samples. (a) Representative immunohistoche-mical stainings show the presence of NHE-1 in human esophageal samples. Scale bar represents 100 µm. (b) Quantification of DAB intensities were calculated, using a semi-quantitative scoring system. Data represent mean ± SEM of 23–25 specimens/3–6 patients each group; a = p = 0.05 vs. normal; b = p = 0.05 vs. non-smoker. BE: Barrett’s esophagus.