Gastroenterology

Gastroenterology

Volume 123, Issue 2, August 2002, Pages 531-541
Gastroenterology

Basic–Alimentary Tract
An alternate pathway of cAMP-stimulated Cl secretion across the NKCC1-null murine duodenum,☆☆

https://doi.org/10.1053/gast.2002.34757Get rights and content

Abstract

Background & Aims: Adenosine 3',5'-cyclic monophosphate (cAMP)-stimulated anion secretion across the duodenal epithelium requires the cystic fibrosis transmembrane conductance regulator (CFTR) in the apical membrane and anion uptake proteins in the basolateral membrane. NKCC1, the epithelial Na+/K+/2Cl cotransporter, is the major protein responsible for Cl uptake. In this study, we evaluate the role of NKCC1 in determining the relative rates of transepithelial Cl and HCO3 secretion during cAMP stimulation of the duodenum. Methods: Bicarbonate and chloride secretion across duodenal mucosa was measured in Ussing chambers by pH stat and 36Cl flux methods using mice with either gene-targeted deletion of NKCC1 (NKCC1−/−) or bumetanide blockade of NKCC1. Results: Total anion secretion stimulated by forskolin treatment of NKCC1-null duodenum resulted from approximately equivalent rates of electrogenic chloride, electrogenic bicarbonate, and electroneutral bicarbonate secretion. Evaluation of the alternate chloride secretory pathway indicated chloride uptake by a basolateral membrane anion exchange process with characteristics consistent with the anion exchanger isoform AE2. Conclusions: Chloride uptake by basolateral anion exchanger activity (AE2) supports intracellular cAMP–stimulated chloride secretion in the NKCC1-null duodenum. A model for the alternate chloride secretion pathway is proposed whereby chloride uptake via AE2 is coupled to basolateral NaHCO3 cotransport to support CFTR-mediated chloride and bicarbonate secretion.

GASTROENTEROLOGY 2002;123:531-541

Section snippets

Animals

Mice with gene-targeted disruption (knockout) of NKCC1 (locus Slc12a2) were developed as previously described.9 At 3–6 weeks of age, offspring were genotyped using primers specific for murine NKCC1 and the disrupted allele. Age-matched NKCC1 (+/+) and NKCC1 (−/−) siblings 2–5 months of age were used for experimentation. The mice were fed a standard laboratory chow (Formulab 5008 Rodent Chow; Ralston Purina, St. Louis, MO) and water ad libitum. Before each experiment, the mice were fasted

pH stat studies

The JsmHCO3 and Isc were measured over 3 consecutive 30-minute periods (basal, forskolin, and forskolin + bumetanide) in NKCC1+/+ and NKCC1−/− duodena. The normalized Isc was converted to μeq/cm2 · h for comparison with the JsmHCO3. In NKCC1+/+ duodenum (Figure 1A), both Isc and JsmHCO3 showed stable rates throughout the basal period (period 1).

. Sequential effects of forskolin and bumetanide on Isc (●) and JsmHCO3 (○) across (A) NKCC1+/+ and (B) NKCC1−/− duodena (n = 6 each). At the arrows,

Discussion

The anion secretory response to stimulation of intracellular cyclic nucleotides in intestinal epithelia involves the combined efflux of Cl and HCO3 ions across the apical membrane by electrogenic processes requiring the anion channel function of CFTR.4 As predicted from the model developed by Field and others,28 completion of the current pathway for sustained Cl secretion necessitates a process of Cl uptake across the basolateral membrane of epithelial cells. Molecular cloning, biochemical,

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    Address requests for reprints to: Lane L. Clarke, D.V.M., Ph.D., 324D Dalton Cardiovascular Research Center, Research Park Drive, University of Missouri-Columbia, Columbia, Missouri 65211. e-mail: [email protected]; fax: (573) 884-4232.

    ☆☆

    Supported by grants RO1 DK48816 and RO1 DK50594 from the National Institutes of Health.

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