Vasoactive intestinal peptide, forskolin, and genistein increase apical CFTR trafficking in the rectal gland of the spiny dogfish, Squalus acanthias. Acute regulation of CFTR trafficking in an intact epithelium.

Abstract

Defective trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR) is the most common cause of cystic fibrosis. In chloride-secreting epithelia, it is well established that CFTR localizes to intracellular organelles and to apical membranes. However, it is controversial whether secretagogues regulate the trafficking of CFTR. To investigate whether acute hormonal stimulation of chloride secretion is coupled to the trafficking of CFTR, we used the intact shark rectal gland, a model tissue in which salt secretion is dynamically regulated and both chloride secretion and cellular CFTR immunofluorescence can be quantified in parallel. In rectal glands perfused under basal conditions without secretagogues, Cl- secretion was 151+/-65 microeq/h/g. Vasoactive intestinal peptide (VIP), forskolin, and genistein led to 10-, 6-, and 4-fold increases in Cl- secretion. In basal glands, quantitative confocal microscopy revealed CFTR immunofluorescence extending from the apical membrane deeply into the cell (7.28+/-0.35 micron). During stimulation with secretagogues, apical extension of CFTR immunofluorescence into the cell was reduced significantly to 3.24+/-0.08 micron by VIP, 4.08+/-0.13 by forskolin, and 3.19+/-0.1 by genistein (P < 0.001). Moreover, the peak intensity of CFTR fluorescence shifted towards the apical membrane (peak fluorescence 2.5+/-0.13 micron basal vs. 1.51+/-0.06, 1.77+/-0.1, and 1.38+/-0.05 for VIP, forskolin, and genistein; all P < 0.001). The increase in both Cl- secretion and apical CFTR trafficking reversed to basal values after removal of VIP. These data provide the first quantitative morphological evidence for acute hormonal regulation of CFTR trafficking in an intact epithelial tissue.

The Short Apical Membrane Half-life of Rescued ΔF508-Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Results from Accelerated Endocytosis of ΔF508-CFTR in Polarized Human Airway Epithelial Cells

“…The decreased apical membrane expression of WT-CFTR and ⌬F508-CFTR in CFBE41oϪ cells is consistent with the view that Rab11a facilitates the endocytic recycling of WT-CFTR and rescued ⌬F508-CFTR in polarized human airway epithelial cells. Endocytosis and endocytic recycling determine, at least in part, the expression of CFTR in the apical membrane (28,81,82). Thus, inhibition of the endocytic recycling of ⌬F508-CFTR by siRNA-mediated silencing of Rab11a together with increased endocytosis of ⌬F508-CFTR should result in lower apical membrane expression of ⌬F508-CFTR compared with WT-CFTR.…”

Marine Organism Cell Biology and Regulatory Sequence Discoveryin Comparative Functional Genomics

“…They exhibit fundamental vertebrate characteristics, including a recombinatorial immune system (Hinds and Litman 1986;Adelman et al 2004), and are also the oldest existing vertebrates with circulatory system-related signaling molecules and receptors such as platelet-derived growth factor and adenosine receptors. The specialized rectal gland of Squalus acanthias, the spiny dogfish shark, has greatly facilitated the study of cystic fibrosis, sodium and chloride secretion (Devor et al 1995;Lehrich et al 1995;Forrest 1996;Henson et al 1997;Lehrich et al 1998;Silva et al 1999;Aller et al 1999;Greger et al 1999;Waldegger et al 1999;Ke et al 2002;Yang et al 2002). Unlike many primary cell cultures that dedifferentiate and lose transport polarity immediately after isolation, primary cultures of shark rectal gland tubular cells maintain fully differentiated function and expression of all known receptors, transporters, ion channels and signal transduction pathways in vitro (Valentich and Forrest 1991;Devor et al 1995;Lehrich et al 1995;Aller et al 1999;Greger et al 1999;Waldegger et al 1999;Ke et al 2002;Yang et al 2002;Mattingly et al 2004b).…”

Rapid hormonal regulation of N-acetylglucosamine transferase I

“…Cyclic nucleotides have been shown to be involved in the translocation of several receptors and transducing molecules; for example, cAMP has been implicated in the nerve growth factor receptor (Meyer-Franke et al 1998), the water channel aquaporin-2 (Fushimi et al 1997), an epithelial chloride channel (Shintani & Marunaka 1996) and the cystic fibrosis transmembrane conductance regulator (Lehrich et al 1998). Furthermore, cGMP is involved in the membrane recruitment of the dopamine receptor (Holtbäck et al 1999).…”