title: Dynamic and differential regulation of NKCC1 by calcium and cAMP in the native human colonic epithelium.
abstract: The capacity of the intestine to secrete fluid is dependent on the basolateral Na(+)-K(+)-2Cl(-) co-transporter (NKCC1). Given that cAMP and Ca(2+) signals promote sustained and transient episodes of fluid secretion, respectively, this study investigated the differential regulation of functional NKCC1 membrane expression in the native human colonic epithelium. Tissue sections and colonic crypts were obtained from sigmoid rectal biopsy tissue samples. Cellular location of NKCC1, Na(+)-K(+)-ATPase, M3 muscarinic acetylcholine receptor (M(3)AChR) and lysosomes was examined by immunolabelling techniques. NKCC1 activity (i.e. bumetanide-sensitive uptake), intracellular Ca(2+) and cell volume were assessed by 2',7'-bis(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF), Fura-2 and differential interference contrast/calcein imaging. Unstimulated NKCC1 was expressed on basolateral membranes and exhibited a topological expression gradient, predominant at the crypt base. Cholinergic Ca(2+) signals initiated at the crypt base and spread along the crypt axis. In response, NKCC1 underwent a Ca(2+)-dependent 4 h cycle of recruitment to basolateral membranes, activation, internalization, degradation and re-expression. Internalization was prevented by the epidermal growth factor receptor kinase inhibitor tyrphostin-AG1478, and re-expression was prohibited by the protein synthesis inhibitor cylcoheximide; the lysosome inhibitor chloroquine promoted accumulation of NKCC1 vesicles. NKCC1 internalization and re-expression were accompanied by secretory volume decrease and bumetanide-sensitive regulatory volume increase, respectively. In contrast, forskolin (i.e. cAMP elevation)-stimulated NKCC1 activity was sustained, and membrane expression and cell volume remained constant. Co-stimulation with forskolin and acetylcholine promoted dramatic recruitment of NKCC1 to basolateral membranes and prolonged the cycle of co-transporter activation, internalization and re-expression. In conclusion, persistent NKCC1 activation by cAMP is constrained by a Ca(2+)-dependent cycle of co-transporter internalization, degradation and re-expression; this is a novel mechanism to limit intestinal fluid loss.
authors.affiliation: School of Biological Sciences, University of East Anglia, Norwich, Norfolk NR4 7TJ, UK.
authors.forename: Amy
authors.initials: A
authors.lastname: Reynolds
authors.forename: Alyson
authors.initials: A
authors.lastname: Parris
authors.forename: Luke A
authors.initials: LA
authors.lastname: Evans
authors.forename: Susanne
authors.initials: S
authors.lastname: Lindqvist
authors.forename: Paul
authors.initials: P
authors.lastname: Sharp
authors.forename: Michael
authors.initials: M
authors.lastname: Lewis
authors.forename: Richard
authors.initials: R
authors.lastname: Tighe
authors.forename: Mark R
authors.initials: MR
authors.lastname: Williams
mesh_terms: D000109:Acetylcholine / Q000494:pharmacology; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D002118:Calcium / Q000502:physiology*; D020013:Calcium Signaling; D002738:Chloroquine / Q000494:pharmacology; D018678:Cholinergic Agents / Q000494:pharmacology; D005576:Colforsin / Q000494:pharmacology; D003106:Colon / Q000378:metabolism*; D000242:Cyclic AMP / Q000502:physiology*; D066246:ErbB Receptors / Q000037:antagonists & inhibitors; D006801:Humans; D007413:Intestinal Mucosa / Q000187:drug effects / Q000378:metabolism*; D008247:Lysosomes / Q000378:metabolism; D008875:Middle Aged; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011799:Quinazolines; D043587:Receptor, Muscarinic M3; D028021:Sodium-Potassium-Chloride Symporters / Q000096:biosynthesis / Q000378:metabolism*; D064506:Solute Carrier Family 12, Member 2; D014018:Tissue Distribution; D020032:Tyrphostins / Q000494:pharmacology
chemical_list: D018678:Cholinergic Agents; D011500:Protein Synthesis Inhibitors; D011799:Quinazolines; D043587:Receptor, Muscarinic M3; C578877:SLC12A2 protein, human; D028021:Sodium-Potassium-Chloride Symporters; D064506:Solute Carrier Family 12, Member 2; D020032:Tyrphostins; C101044:RTKI cpd; D005576:Colforsin; D002738:Chloroquine; D000242:Cyclic AMP; D066246:ErbB Receptors; D000109:Acetylcholine; D002118:Calcium