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¹ School of Biological Sciences, University of East Anglia, Norwich, Norfolk NR4 7TJ, UK
² Department of General Surgery, Norfolk and Norwich University Hospital, Norwich, Norfolk NR4 7UY, UK
³ Department of Gastroenterology, Norfolk and Norwich University Hospital, Norwich, Norfolk NR4 7UY, UK

The capacity of the intestine to secrete fluid is dependent on the basolateral Na⁺–K⁺–2Cl^– co-transporter (NKCC1). Given that cAMP and Ca²⁺ 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 (M₃AChR) and lysosomes was examined by immunolabelling techniques. NKCC1 activity (i.e. bumetanide-sensitive uptake), intracellular Ca²⁺ 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 Ca²⁺ signals initiated at the crypt base and spread along the crypt axis. In response, NKCC1 underwent a Ca²⁺-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 Ca²⁺-dependent cycle of co-transporter internalization, degradation and re-expression; this is a novel mechanism to limit intestinal fluid loss.

(Received 6 February 2007; accepted after revision 26 April 2007; first published online 5 May 2007)
Corresponding author M. Williams: School of Biological Sciences, University of East Anglia, Norwich, Norfolk NR4 7TJ, UK. Email: m.r.williams{at}uea.ac.uk

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