Yung-Lung Chang1, Kai Ren Jian1, Chun-Shu Lin2, Hsing-Won Wang3,4, Shao-Cheng Liu3. 1. Department of Biochemistry, National Defense Medical Center, Taipei, Taiwan, Republic of China. 2. Department of Radiation Oncology, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, Republic of China. 3. Department of Otolaryngology-Head and Neck Surgery, Tri-Service General Hospital, National Defense Medical Center. 4. Department of Otolaryngology-Head and Neck Surgery, Shuang Ho Hospital, Taipei, Taiwan, Republic of China.
Abstract
BACKGROUND: Cholinergic stimulation plays a major role in inflammatory airway diseases. However, its role in airway surface liquid homeostasis and aquaporin 5 (AQP5) regulation remains unclear. In this study we sought to determine the effects of methacholine and dexamethasone on AQP5 expression in human nasal epithelial cells (HNEpC). METHODS: HNEpC were cultured with methacholine or dexamethasone at 4 concentrations in vitro. The subcellular distribution of AQP5 was explored using immunocytochemistry. The pharmacologic effects of methacholine and dexamethasone on the expression of the phosphorylation of cyclic adenosine monophosphate-responsive element binding protein (p-CREB), AQP5, and nuclear factor-kappaB (NF-κB) were examined using Western blotting. RESULTS: AQP5 was found to be located in cell membrane and cytoplasm and present in every group without a statistically significant difference. Methacholine inhibited expression of AQP5 and p-CREB in HNEpC, whereas dexamethasone increased these protein levels dose-dependently in a statistically significant manner. In turn, HNEpC treated with methacholine and dexamethasone showed the same trends as those intervened separately with these 2 drugs. Moreover, dexamethasone had the ability to reverse the inhibitory effect of methacholine. Western blotting revealed that, after incubation with 10-4 mol/L methacholine, NF-κB increased significantly, by 186.67%, compared with the untreated control group. Again, such an increase could be significantly reversed after dexamethasone treatment. CONCLUSION: NF-κB activation is important for inhibition of p-CREB/AQP5 expression after methacholine intervention, and dexamethasone adjusts it to the opposite side. This observation could provide additional insight into the anti-inflammatory effects of glucocorticoids that contribute to maintaining airway surface liquid and mucosal defense.
BACKGROUND: Cholinergic stimulation plays a major role in inflammatory airway diseases. However, its role in airway surface liquid homeostasis and aquaporin 5 (AQP5) regulation remains unclear. In this study we sought to determine the effects of methacholine and dexamethasone on AQP5 expression in human nasal epithelial cells (HNEpC). METHODS: HNEpC were cultured with methacholine or dexamethasone at 4 concentrations in vitro. The subcellular distribution of AQP5 was explored using immunocytochemistry. The pharmacologic effects of methacholine and dexamethasone on the expression of the phosphorylation of cyclic adenosine monophosphate-responsive element binding protein (p-CREB), AQP5, and nuclear factor-kappaB (NF-κB) were examined using Western blotting. RESULTS:AQP5 was found to be located in cell membrane and cytoplasm and present in every group without a statistically significant difference. Methacholine inhibited expression of AQP5 and p-CREB in HNEpC, whereas dexamethasone increased these protein levels dose-dependently in a statistically significant manner. In turn, HNEpC treated with methacholine and dexamethasone showed the same trends as those intervened separately with these 2 drugs. Moreover, dexamethasone had the ability to reverse the inhibitory effect of methacholine. Western blotting revealed that, after incubation with 10-4 mol/L methacholine, NF-κB increased significantly, by 186.67%, compared with the untreated control group. Again, such an increase could be significantly reversed after dexamethasone treatment. CONCLUSION: NF-κB activation is important for inhibition of p-CREB/AQP5 expression after methacholine intervention, and dexamethasone adjusts it to the opposite side. This observation could provide additional insight into the anti-inflammatory effects of glucocorticoids that contribute to maintaining airway surface liquid and mucosal defense.