Clara cell impact in air-side activation of CFTR in small pulmonary airways

Abstract

The Clara cells are nonciliated, nonmucous, secretory cells containing characteristic peptidergic granules; they constitute up to 80% of the epithelial cell population of the distal airways. Despite this exposed histotopology and abundance within the terminal airways where fluid secretion is of pivotal importance, the functional role of the Clara cells remained poorly understood. At the transcriptional, translational, and cellular levels, we provide evidence that the Clara cells are well equipped with the bioactive peptide guanylin and proteins of the cGMP-signaling system including guanylate cyclase C, cGMPdependent protein kinase II, and cystic fibrosis transmembrane conductance regulator (CFTR) together with the two CFTR scaffolding proteins EBP50͞NHERF and E3KARP͞NHERF-2 that are essential for proper function of CFTR. Guanylin was localized to secretory granules underneath the apical membrane of Clara cells and was, in addition, detected in high concentrations in bronchoalveolar lavage fluid, predicting release of the peptide luminally into the bronchiolar airways. On the other hand, the guanylin-receptor guanylate cyclase C, CFTR, and proteins linked to CFTR activation and function were all confined to the adluminal membrane of Clara cells, implicating an intriguing air-side route of action of guanylin. Whole-cell patch-clamp recordings in the Clara cell line H441 revealed that guanylin activates CFTR Cl ؊ conductance via the cGMP but not the cAMP-signaling pathway. Hence, in the critical location of distal airways in situ, the Clara cells may play the outstanding role of CFTR-dependent regulation of epithelial electrolyte͞water secretion through a sophisticated paracrine͞luminocrine mode of guanylin-induced CFTR activation.guanylin ͉ guanylate cyclase c ͉ lung ͉ cystic fibrosis ͉ electrolyte secretion

The regulation of selective and nonselective Na+ conductances in H441 human airway epithelial cells

“…The insulin/PI3K-induced G Cl therefore resembles an anion-selective conductance recently identified in rat cortical lens fiber cells (55). Although it has been suggested that H441 cells express significant amounts of CFTR (30,33), these channels are characteristically less permeable to I Ϫ than to Cl Ϫ , and so the present data, in common with earlier work (56), suggest that CFTR does not contribute significantly to G Cl under the present conditions. Further evidence of this came from that fact that CFTR-172, which blocks CFTR with a reasonable degree of selectivity (36), had no effect on the conductive properties of insulin-treated cells.…”

Characterization of Wild-Type and ΔF508 Cystic Fibrosis Transmembrane Regulator in Human Respiratory Epithelia

“…We localized ezrin and EBP50 to the apical PM of human airway epithelial cells (Short et al, 1998;Mohler et al, 1999;Ingraffea et al, 2002;Kulaksiz et al, 2002). Both proteins colocalized to the microvillus domain of epithelial cells in nasal, tracheal, bronchial, and bronchiolar superficial epithelia and were expressed at equivalent levels in CF and normal epithelia.…”

The iron-regulatory peptide hormone hepcidin: expression and cellular localization in the mammalian kidney

“…Proteins from human, mouse and rat kidney and liver, as well as from human urine (50 ml for each experiment) were extracted according to the published protocols (Kulaksiz et al 2002a(Kulaksiz et al ,b, 2004. Following electrophoresis, proteins were transferred onto hydrophobic polyvinylidene fluoride-based membranes (Pall, Portsmouth, Hants, UK) by semidry blotting.…”

“…Paraffin sections (4-5 µm) were immunostained for hepcidin (antibodies EG (1) by the avidin-biotin-peroxidase complex technique as detailed elsewhere (Kulaksiz et al 2002a(Kulaksiz et al ,b, 2004.…”