Salt stress increases abundance and glycosylation of CFTR localized at apical surfaces of salt gland secretory cells.
Osmotic stress elicits hypertonic NaCl secretion and promotes structural and biochemical differentiation in avian salt glands. In addition to cholinergic control, Cl- secretion is stimulated by vasoactive intestinal peptide (VIP), suggesting that the cystic fibrosis transmembrane conductance regulator (CFTR) may be present and that its expression may be regulated by chronic salt stress. Anion efflux, assayed by 6-methoxy-N-(3-sulfopropyl)quinolinium fluorescence changes in single cells, was stimulated by VIP or 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate. Immunoblots with a COOH-terminal peptide antibody to human CFTR revealed approximately 170- and approximately 180-kDa bands in lysates from control and salt-stressed glands, respectively. Both variants reduced to approximately 140 kDa after N-glycanase digestion and gave identical tryptic phosphopeptide maps after immunoprecipitation and phosphorylation by protein kinase A. CFTR was localized to apical membranes by immunofluorescence and, additionally, to subapical vesicles by immunoelectron microscopy. Salt stress induced an approximately twofold increase in CFTR abundance/cell protein (approximately 5-fold/cell) and intensified apical membrane immunofluorescence. For comparison, Na+ pump expression increased approximately fourfold per cell protein with little change in actin. Thus differentiation induced by salt stress is accompanied by alteration in CFTR abundance and glycosylation. Upregulation of CFTR likely contributes to increased efficiency of Cl- secretion.
Ernst
S A
SA
Department of Anatomy and Cell Biology, University of Michigan, Ann Arbor 48109.
Crawford
K M
KM
Post
M A
MA
Cohn
J A
JA
D000581:Amidohydrolases / Q000378:metabolism; D000818:Animals; D000831:Animals, Newborn; D002462:Cell Membrane / Q000378:metabolism; D018118:Chloride Channels / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D004372:Ducks; D006031:Glycosylation; D007150:Immunohistochemistry; D008565:Membrane Proteins / Q000378:metabolism*; D009997:Osmotic Pressure; D010449:Peptide Mapping; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D010750:Phosphoproteins / Q000737:chemistry; D012491:Salt Gland / Q000166:cytology / Q000378:metabolism*; D012965:Sodium Chloride / Q000494:pharmacology*; D014018:Tissue Distribution
D018118:Chloride Channels; D008565:Membrane Proteins; D010750:Phosphoproteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D012965:Sodium Chloride; D017868:Cyclic AMP-Dependent Protein Kinases; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase