EGTA treatment of human airways in vitro unmasks M1/MUC5AC mucin in submucosal glands

Mucin staining can be used to evaluate secretory activity of human airways. However, mucin epitopes may be masked by physicochemical properties of the secretions. The aim of this investigation was to examine the effects of th e calcium chelator, ethyleneglycol-bis-(beta -aminoethylether)-N, N, N ', N ' -tetraacetic acid (EGTA) on the detection of M1/MUC5AC mucin in isolated human bronchial preparations. Immunohistochemical investigation and immunoradiometric assays with anti-Ni t monoclonal antibodies (Mabs) were used to detect M1/MUC5AC mucin derived from bronchial preparations with an intact surface epithelium, or in tissue s where the epithelium had been removed (rubbed preparations). The Mabs labelled both epithelial goblet cells and submucosal glandular cel ls in EGTA (4 mM)-exposed bronchial preparations, while only goblet cells w ere stained in EGTA (0.4 mM)-exposed tissues. The quantities of M1/MUC5AC m ucin detected in either the bronchial fluids derived from EGTA (4 mM)-expos ed intact and rubbed preparations or in bronchial fluids treated with EGTA (4 mM) were significantly increased by two-fold when compared with untreate d control values (p <0.001). In addition, lactate dehydrogenase (LDH) activ ity and protein measurements were unaltered during exposure of human airway s to EGTA (4 mM) suggesting that this treatment did not affect tissue viabi lity. These results provide evidence that ethyleneglycol-bis-(beta -aminoethyleth er)-N. N, N ' N ' -tetraacetic acid (4 mM) facilitates the detection of M1/ MUC5AC mucin by altering the physicochemical properties of respiratory muci n, thereby exposing epitopes; with which anti-Ml monoclonal antibodies are reactive. This will allow more accurate measurement of secretory activity i n human airways in vitro.