Permeabilization via the P2X7 purinoreceptor reveals the presence of a Ca2+ -activated Cl- conductance in the apical membrane of murine tracheal epithelial cells

Calcium-activated Cl- secretion is an important modulator of regulated ion transport in murine airway epithelium and is mediated by an unidentified Ca2+-stimulated Cl- channel. We have transfected immortalized murine tracheal epithelial cells with the cDNA encoding the permeabilizing P2X7 purinoreceptor (P2X7-R) to selectively permeabilize the basolateral membrane and thereby isolate the apical membrane Ca2+-activated Cl- current. In P2X7-R-permeabilized cells, we have demonstrated that UTP stimulates a Cl- current across the apical membrane of CF and normal murine tracheal epithelial cells. The magnitude of the UTP-stimulated current was significantly greater in CF than in normal cells. Ion substitution studies demonstrated that the current exhibited a permselectivity sequence of Cl- > I- > Br- > gluconate-. We have also determined a rank order of potency for putative Cl- channel blockers: niflumic acid ≥ 5-nitro-2-(3-phenylpropylamino)benzoic acid > 4,4'-diisothiocyanostilbene-2,2'-disulfonate > glybenclamide >> diphenlyamine-2-carboxylate, tamoxifen, and p-tetra-sulfonato-tetra-methoxy-calix[4]arene. Complete characterization of this current and the corresponding single channel properties could lead to the development of a new therapy to correct the defective airway surface liquid in cystic fibrosis patients

Nucleotide regulation of goblet cells in human airway epithelial explants: normal exocytosis in cystic fibrosis.

The regulation of mucin secretion by airway goblet cells is poorly understood and the receptor-based regulatory mechanisms have not been described in human airways. In the present study, we report that extracellular triphosphate nucleotides regulate the rate of granule release from goblet cells in both normal and cystic fibrosis (CF) airway epithelial explants. Explants isolated from nasal and tracheobronchial tissues were mounted in perfusion chambers and the secretory activity was assessed by videomicroscopic determination of degranulation in single goblet cells and by ELISA determination of mucins secreted into the mucosal perfusate. Baseline degranulation was measured at 0.05 degranulation events (DE)/min. In normal goblet cells, mucosal ATP (10(-4) M, n = 17) induced a biphasic secretory response comprising 29.1 +/- 4.9 DE during the first 5 min, with an initial rate of 118.2 +/- 10.2 DE/min. Mucosal UTP (10(-4) M, n = 9) induced a similar response to ATP (initial rate: 89.2 +/- 23.9 DE/min, 17.9 +/- 5.1 DE in 5 min), but mucosal 2-MeSATP was not an effective agonist (initial rate: 1.5 +/- 1.4 DE/min, 2.3 +/- 0.5 DE in 5 min). Determination of mucins by ELISA confirmed that both ATP and UTP induced similar secretory responses but that 2-MeSATP was not effective. In CF explants, mucosal UTP (10(-4) M, n = 6) induced similar responses to those observed in normal tissues (initial rate: 82.5 +/- 27.5 DE/min, 18.8 +/- 4.1 DE in 5 min). We conclude that human nasal and tracheobronchial goblet cells are stimulated by mucosal nucleotides, probably via a 5'-nucleotide receptor, and that this response is unaffected by CF