Multiple intracellular pathways for regulation of chloride secretion in cultured pig tracheal submucosal gland cells. A.-L. Zhang, G.M. Roomans. #ERS Journals Ltd 1999. ABSTRACT: Tracheal submucosal glands are of great relative importance in the secretion of chloride and water to the airway lumen. This study aimed to examine whether the cystic fibrosis transmembrane conductance regulator (CFTR) is involved in cyclic adenosine monophosphate (cAMP) or Ca 2+ -activated Cl -secretion. Regulation of Cl -secretion in cell cultures derived from pig tracheal submucosal gland acini was investigated by X-ray microanalysis. With or without preincubation with CFTR antisense oligodeoxynucleotide (5 mM).A significant decrease in cellular Cl and K concentration was induced by 5 mM 8-bromo-adenosine 3': 5'-cyclic monophosphate (8-bromo-cAMP), 3 mM calcium ionophore ionomycin, 200 mM 5'-uridine triphosphate (UTP) and 200 mM 5'-adenosine triphosphate (ATP), respectively. The decrease in cellular Cl content was significantly inhibited by the Cl -channel blocker 5-nitro-2-(3-phenylpropyl-amino)-benzoic acid (NPPB; 50 mm). Preincubation of the cells with CFTR antisense oligodeoxynucleotide significantly inhibited the 8-bromo-cAMP-induced decrease in Cl, whereas CFTR sense oligodeoxynucleotide had no effect. The effects of ionomycin, ATP or UTP were not blocked by either CFTR antisense oligodeoxynucleotide or CFTR sense oligodeoxynueleotide. To measure the cytosolic free calcium concentration ([Ca 2+ ] i ) the cells grown on glass coverslips were loaded with fura-2 tetraoxymethylester (fura-2 AM; 5 mM). The [Ca 2+ ] i was measured as the fluorescence ratio of emission (340/380 nm). Ionomycin (3 mM) caused a rapid increase in [Ca 2+ ] i followed by a sustained plateau, but 8-bromo-cAMP had a more complex effect on [Ca 2+ ] i . Exposure to ATP or UTP caused a rapid increase in [Ca 2+ ] i followed by a decrease.In conclusion, cystic adenosine monophosphate and ionomycin induced Cl -secretion through different intracellular pathways. Adenosine triphosphate and uridine triphosphate also induced Cl -secretion probably with Ca 2+ as an intracellular messenger. The cystic fibrosis transmembrane conductance regulator is not involved in Cl -secretion activated by extracellular adenosine triphosphate and uridine triphosphate. Eur Respir J 1999; 13: 571±576.

The submucosal glands of the airway may contribute more to the airway fluid than the surface epithelium. The cellular mechanisms underlying the regulation of electrolyte and water transport in airway submucosal glands are, however, still poorly understood. Therefore, we attempted to establish a cell culture system to facilitate study of this regulation.Acinar cells were isolated by enzymatic disaggregation from pig tracheal submucosal tissue and cultured on a plastic substrate coated with human placental collagen. The fine structure of the cells in confluent culture was studied by conventional transmission electron microscopy. The elemental content in resting cells and stimulated cells grown on a permeable substrate was studied by X-ray microanalysis.The cultured cells retained structural characteristics (microvilli, secretory granules and desmosomes) of in situ epithelia. The total intracellular Cl and K concentrations significantly decreased after stimulation with the cholinergic agonist acetylcholine, the predominantly alpha-adrenergic agonist norepinephrine, or the beta-adrenergic agonist isoproterenol. Both ionomycin and 8-bromoadenosine 3':5'-cyclic monophosphate (8-bromo-cAMP) caused a marked decrease of the intracellular Cl, K and Na concentrations. Cl -secretion induced by acetylcholine was inhibited by Cl -channel blockers anthracene-9-carboxylic acid and 5-nitro-2-(3-phenylpropyl-amino)-benzoic acid (NPPB), but Cl -efflux induced by 8-bromocAMP was blocked only by NPPB. The intracellular Cl and Na content significantly increased and the cellular K content markedly decreased after treatment with ouabain.These results indicate that the cultured acinar cells maintained the structural and functional characteristics of in situ tissue and that this system is suitable for studying aspects of ion and water transport by the airway submucosal gland cells.

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