Down-regulated in adenoma Cl/HCO3 exchanger couples with Na/H exchanger 3 for NaCl absorption in murine small intestine

Abstract

Background & aims: Electroneutral NaCl absorption across small intestine contributes importantly to systemic fluid balance. Disturbances in this process occur in both obstructive and diarrheal diseases, eg, cystic fibrosis, secretory diarrhea. NaCl absorption involves coupling of Cl(-)/HCO(3)(-) exchanger(s) primarily with Na(+)/H(+) exchanger 3 (Nhe3) at the apical membrane of intestinal epithelia. Identity of the coupling Cl(-)/HCO(3)(-) exchanger(s) was investigated using mice with gene-targeted knockout (KO) of Cl(-)/HCO(3)(-) exchangers: Slc26a3, down-regulated in adenoma (Dra) or Slc26a6, putative anion transporter-1 (Pat-1).

Methods: Intracellular pH (pH(i)) of intact jejunal villous epithelium was measured by ratiometric microfluoroscopy. Ussing chambers were used to measure transepithelial (22)Na(36)Cl flux across murine jejunum, a site of electroneutral NaCl absorption. Expression was estimated using immunofluorescence and quantitative polymerase chain reaction.

Results: Basal pH(i) of DraKO epithelium, but not Pat-1KO epithelium, was alkaline, whereas pH(i) in the Nhe3KO was acidic relative to wild-type. Altered pH(i) was associated with robust Na(+)/H(+) and Cl(-)/HCO(3)(-) exchange activity in the DraKO and Nhe3KO villous epithelium, respectively. Contrary to genetic ablation, pharmacologic inhibition of Nhe3 in wild-type did not alter pH(i) but coordinately inhibited Dra. Flux studies revealed that Cl(-) absorption was essentially abolished (>80%) in the DraKO and little changed (<20%) in the Pat-1KO jejunum. Net Na(+) absorption was unaffected. Immunofluorescence demonstrated modest Dra expression in the jejunum relative to large intestine. Functional and expression studies did not indicate compensatory changes in relevant transporters.

Conclusions: These studies provide functional evidence that Dra is the major Cl(-)/HCO(3)(-) exchanger coupled with Nhe3 for electroneutral NaCl absorption across mammalian small intestine.

Figure 1

Intracellular pH (pH_i) in intact villous epithelium. Left panel: basal pH_i in WT, Pat-1 KO, Dra KO, and Nhe3 KO intestine. Right panel: effect of inhibiting apical membrane Na⁺/H⁺ exchange with 100 μmol/L EIPA or Cl⁻/HCO₃⁻ exchange with 100 μmol/L niflumic acid (NFA) in WT epithelium. *Experimental conditions accentuate the effect of apical membrane acid-base transporters (see Results section). ^a,b,cMeans without the same letters are significantly different (n = 4–8 mice).

Figure 2

Functional activities of apical membrane Na⁺/H⁺ and Cl⁻/HCO₃⁻ in intact villous epithelium. (A) Na⁺/H⁺ exchange activity in WT, Dra KO, and Nhe3 KO jejunum as measured by removal and replacement of luminal Na⁺ (representative of 3 mice). (B) Cl⁻/HCO₃⁻ exchange activity in WT, Nhe3 KO, and Dra KO measured by removal and replacement of luminal Cl⁻ (representative of 3 mice).

Figure 3

Basal pH_i and isolated Dra Cl⁻/HCO₃⁻ exchange rates during inhibition of apical membrane Na⁺/H⁺ exchange in Pat-1 KO jejunal villous epithelium. Apical membrane Na⁺/H⁺ (Nhe3) exchange was inhibited with 100 μmol/L EIPA. Control was treated with vehicle (DMSO 0.1%). *Significantly different from control (n = 6–9).

Figure 4

Immunofluorescent localization of Dra (red) and phalloidin (actin, green) in the jejunal and cecal epithelium of WT and Dra KO mice. WT shows immunolocalization of Dra to apical and subapical compartments at the apical membrane that is not present in the Dra KO mice. Expression of immunoreactive Dra is modest in the jejunum villous epithelium relative to the surface epithelium of the cecum (representative of 4 experiments). Bars = 25 μm; arrows indicate apical membrane.