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CELLULAR AND MOLECULAR

Vasoactive Intestinal Peptide Increases Cystic Fibrosis Transmembrane Conductance Regulator Levels in the Apical Membrane of Calu-3 Cells through a Protein Kinase C-Dependent Mechanism

Department of Physiology and Biophysics, Dalhousie University, Halifax, Nova Scotia, Canada (F.C., V.C.); and Department of Physiology, McGill University, Montréal, Quebec, Canada (M.E.L., J.W.H.)

Noncholinergic neurons contribute to innate airway defenses by releasing vasoactive intestinal peptides (VIP), which stimulates the submucosal glands to produce a bicarbonate-rich fluid containing mucins and antimicrobial factors. VIP elevates cAMP and activates cystic fibrosis transmembrane conductance regulator (CFTR) channels; however, its effects on surface expression have not been investigated. We studied CFTR levels in the apical membrane of polarized Calu-3 cell monolayers, a widely used model for submucosal gland serous cells. Biotinylation during VIP exposure revealed a significant increase in apical CFTR within 10 min, which reached a maximal 3.3-fold increase after 30 min. Total CFTR content of cell lysates was not altered during this time period; therefore, the increase in surface CFTR reflects redistribution from intracellular pools. Internalization assays revealed that apical accumulation was due, at least in part, to a reduction in the rate of CFTR endocytosis. VIP-induced accumulation of apical CFTR was mimicked by phorbol ester but not by forskolin, and it was blocked by the protein kinase (PK)C inhibitors bisindolylmaleimide X (BisX) or chelerythrine chloride but not by the PKA inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H89). Increases in surface expression were paralleled by enhanced iodide effluxes during cAMP stimulation. BisX inhibition of VIP responses was abrogated when monolayers were pretreated with tannic acid to inhibit endosome recycling. Thus, PKC increases the surface expression of CFTR channels in addition to potentiating their responsiveness to PKA phosphorylation. Integrated regulation through multiple signaling pathways may be a common feature of VIP and other physiological secretagogues.

Address correspondence to: Dr. Valérie Chappe, Department of Physiology and Biophysics, 5850 College Street, Halifax, Nova Scotia, Canada B3H 1X5. E-mail: valerie.chappe{at}dal.ca

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