Ion transport in cultured pig tracheal submucosal gland acinar cells studied by X-ray microanalysis

Abstract

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.

Multiple intracellular pathways for regulation of chloride secretion in cultured pig tracheal submucosal gland cells

“…The observation in this study that [Ca 2+ ] i clearly increased after ionomycin addition indicates that a Ca 2+ -activated pathway of Cl ± secretion exists in the cultured cells. Cl ± secretion in cell cultures caused by the b-adrenergic agonist isoproterenol has previously been demonstrated [16]. YAMAYA et al [24] also showed that isoproterenol induced Cl ± secretion in primary culture of human tracheobronchial gland cells, which is in line with the present results because isoproterenol is presumed to act via cAMP as an intracellular messenger.…”

“…Acinar cells were isolated and cultured as previously described [16]. Briefly, submucosal tissue was collected after the surface epithelium was removed by sharp dissection, then digested for 2 h at 378C in digesting medium containing 20 mM HEPES, type IV collagenase (500 U .…”

A procedure to prepare cultured cells in suspension for electron probe X‐ray microanalysis: application to scanning and transmission electron microscopy

“…There is growing interest in the use of cultured cells as experimental models to investigate elemental composition under different physiological and experimental conditions using X‐ray microanalysis ( Wroblewski & Wroblewski, 1993; Warley, 1994; Roomans et al ., 1996 ). Different methods have been developed and various conditions have been used to grow and prepare cultured cells for X‐ray microanalytical studies using electron probe microanalysis ( Abraham et al ., 1985 ), scanning electron microscopy (SEM) ( Zierold & Schäffer, 1988; Hall et al ., 1992 ; von Euler et al ., 1993 ; Borgmann et al ., 1994 ) and scanning transmission electron microscopy (STEM) ( James‐Kracke et al ., 1980 ; Buja et al ., 1985 ; Zierold & Schäffer, 1988; Warley et al ., 1994 ; Hongpaisan et al ., 1996 ; Zhang & Roomans, 1997a). These procedures, however, have been developed fundamentally for cells growing as monolayers on filmed grids, and the cells have generally been analysed as whole mounts and only occasionally as thin cryosections ( Buja et al ., 1985 ; Warley et al ., 1994 ).…”