The Roles Of Aquaporins Protein And Chloride Channel MCLCA3 In Development Of Asthma

In respiratory system, efficient water transport across epithelia and endothelia is essential to maintain normal physiological functions. It is well known that at least four members of aquaporins are expressed in lung and airways, and they are distributed throughout the nasopharynx, airways and lung tissues: AQP1 in microvascular endothelia, AQP3 in basal cells and AQP4 located in apical epithelial cells of airways, and AQP5 mainly in type I alveolar epithelial cells and submucosal gland acini. Previous studies using transgenic knockout mice demonstrated that AQP1and AQP5 provide the principal route for osmotically driven water transport between airspace and capillary compartments; AQP3 and AQP4 faciliate osmotic water transport in airways. In addition, AQP5 deletion remarkably reduces submucosal gland fluid secretion.So far, little is known about the roles of aquaporins in respiratory pathophysiology. There is only one report showing that AQP5-deficient mice manifest significantly increased airway hyperresponsiveness compared with wild type mice upon acetyl choline challenge.In the present study, the roles of aquaporins in asthma were investigated systematically in asthmatic model by using aquaporin knockout mice. The study uncovered important roles of aquaporins in asthma for the first time: compared with wildtype asthmatic mice, asthmatic AQP5-deficient mice manifested more severe mucus hypersecretion, whereas asthmatic AQP4-deficient mice exhibited reduced mucus hypersecretion. Down-regulation of AQP4 was found in asthmatic wildtype mice but not in asthmatic AQP5 deficient mice. These results provide the first evidence that aquaporins are involved in the mucus secretion during asthma. Down-regulation of AQP4 and upregulation of AQP5 may reduce the mucus hypersecretion and alleviate the asthma symptoms.Calcium-activated chloride channels (CACCs) are found as a new chloride channel family. Studies about the protein structure and function of CACC family have been contraversal. mCLCA3 is a member of CACC family in mouse that plays an important role in mucin packaging and hypersecretion during asthma. To study the protein structure and expression of mCLCA3 in asthmatic mouse lung, we expressed an N-terminal 269 amino acid peptide of mCLCA3 in E. coli, purified the peptide to homogeneity and generated rabbit polyclonal antibodies against this peptide. Immunohistochemistry of asthmatic mouse lung using the antibody indicated exclusive mCLCA3 expression in mucin granules of goblet cells in airway surface and lumen. Immunoblot analysis of lavage fluid from asthmatic mouse lung revealed a single 90 kDa protein form of mClCA3. The results demonstrated that the 90 kDa N-terminal peptide, but not the full-length protein or the reported N-terminal 35 kDa cleaved form of mClCA3 is the major functional form involved in the packaging and exocytosis of mucin granules in asthmatic goblet cells. For functional characterization of mCLCA3, we established a stably transfected FRT cell line expressing mCLCA3 and analyzed its chloride channel mfunction. The results do not support mCLCA3 as a chloride channel.