The switch of intestinal Slc26 exchangers from anion absorptive to HCOFormula secretory mode is dependent on CFTR anion channel function

Abstract

CFTR has been recognized to function as both an anion channel and a key regulator of Slc26 anion transporters in heterologous expression systems. Whether this regulatory relationship between CFTR and Slc26 transporters is seen in native intestine, and whether this effect is coupled to CFTR transport function or other features of this protein, has not been studied. The duodena of anesthetized CFTR-, NHE3-, Slc26a6-, and Scl26a3-deficient mice and wild-type (WT) littermates were perfused, and duodenal bicarbonate (HCO(3)(-)) secretion (DBS) and fluid absorptive or secretory rates were measured. The selective NHE3 inhibitor S1611 or genetic ablation of NHE3 significantly reduced fluid absorptive rates and increased DBS. Slc26a6 (PAT1) or Slc26a3 (DRA) ablation reduced the S1611-induced DBS increase and reduced fluid absorptive rates, suggesting that the effect of S1611 or NHE3 ablation on HCO(3)(-) secretion may be an unmasking of Slc26a6- and Slc26a3-mediated Cl(-)/HCO(3)(-) exchange activity. In the absence of CFTR expression or after application of the CFTR(inh)-172, fluid absorptive rates were similar to those of WT, but S1611 induced virtually no increase in DBS, demonstrating that CFTR transport activity, and not just its presence, is required for Slc26-mediated duodenal HCO(3)(-) secretion. A functionally active CFTR is an absolute requirement for Slc26-mediated duodenal HCO(3)(-) secretion, but not for Slc26-mediated fluid absorption, in which these transporters operate in conjunction with the Na(+)/H(+) exchanger NHE3. This suggests that Slc26a6 and Slc26a3 need proton recycling via NHE3 to operate in the Cl(-) absorptive mode and Cl(-) exit via CFTR to operate in the HCO(3)(-) secretory mode.

Fig. 1.

Both pharmacological inhibition (A) and genetic deletion (B) of the Na⁺/H⁺ exchanger isoform NHE3 stimulated duodenal bicarbonate (HCO₃⁻) secretion (DBS) in a Cl⁻-dependent fashion. A: pharmacological inhibition of NHE3 (via S1611) significantly stimulated DBS. B: NHE3-deficient duodenum displayed significantly higher DBS than wild-type (WT) duodenum, and S1611 caused no further stimulation in DBS (C). In both in NHE3-deficient and WT (and in S1611-pretreated, not shown) duodenum, the removal of luminal Cl⁻ caused a strong decrease in DBS, indicating that the Slc26 Cl⁻/HCO₃⁻ exchangers are active in the presence and absence of NHE3. The residual HCO₃⁻ secretion after Cl⁻ removal in NHE3-deficient duodenum reveals Cl⁻-independent HCO₃⁻ secretory pathways (at least in part direct CFTR-mediated HCO₃⁻ secretion, see Supplemental Material, Supplemental Fig. 3) are also present in murine duodenum. Results are means ± SE. *P < 0.05; **P < 0.01; ***P < 0.001, significant difference between 2 groups. #P < 0.05; ##P < 0.01; ###P < 0.001, significant difference compared with basal response. The number of mice (n) euthanized for the experiments is given in parentheses.

Fig. 2.

S1611 does not stimulate electrogenic anion pathways. Isolated duodenal mucosa was placed in classic Ussing-chamber systems, forskolin (10 μM), 16,16-dimethyl PGE₂ (1 μM), and S1611 (20 μM) were applied to the luminal or serosal bath, respectively, and the short-circuit current (I_sc) response was measured. A: time course of the forskolin experiments. A strong I_sc response was induced by forskolin when applied on the serosal side, whereas no response was observed when a similar concentration was applied on the mucosal side. B: net peak I_sc response. S1611 elicited minimal I_sc responses, and this minimal I_sc response was positive when the substance was added to the luminal side (correct side for brush-border membrane NHE3 inhibition). This indicates that the S1611-stimulated HCO₃⁻ secretion does not occur via the activation of an anion conductance. Results are means ± SE. ***P < 0.001, significant difference between serosal and mucosal I_sc response. ###P < 0.001, significant difference between basal and stimulated-state I_sc response within the same group. The number of mice euthanized for the experiments is given in parentheses.

Fig. 3.

Slc26a6 is involved in S1611-stimulated DBS. S1611 (20 μM) caused a significant stimulation of DBS in WT duodenum, and this stimulatory effect was significantly lower in Slc26a6-deficient duodenum. Results are means ± SE. *P < 0.05, significant difference between 2 groups. #P < 0.05; ##P < 0.01; ###P < 0.001, significant difference compared with basal response. The number of mice euthanized for the experiments is given in parentheses.

Fig. 4.

Slc26a3 is involved in S1611-stimulated DBS. S1611 (20 μM) caused a significant stimulation of DBS in WT duodenum, and this stimulatory effect was significantly lower in Slc26a3-deficient duodenum. Results are means ± SE. *P < 0.05; **P < 0.01; ***P < 0.001, significant difference between 2 groups. #P < 0.05; ##P < 0.01; ###P < 0.001, significant difference compared with basal response. The number of mice euthanized for the experiments is given in parentheses.

Fig. 5.

CFTR is involved in S1611-stimulated DBS but is not important for fluid absorption. A: in CFTR-deficient duodenum, basal DBS was significantly lower than in WT duodenum, and no significant S1611-stimulated DBS was observed. This indicates that CFTR expression is essential for Slc26-mediated HCO₃⁻ secretion. B: fluid absorption was not significantly different in CFTR-deficient duodenum, and S1611 inhibited fluid absorption in both WT and CFTR-deficient duodenum. The time course of inhibition of fluid absorption by S1611 was slower in CFTR-deficient than WT duodenum, yet a 60% inhibition was achieved in the second 30 min. Thus the reason for the lack of S1611-mediated stimulation of DBS was a necessity of CFTR for Slc26-mediated HCO₃⁻ secretion, not an inactivity of NHE3 in CFTR-deficient duodenum. Results are means ± SE. *P < 0.05; **P < 0.01; ***P < 0.001, significant difference between 2 groups. #P < 0.05; ##P < 0.01; ###P < 0.001, significant difference compared with basal response. The number of mice euthanized for the experiments is given in parentheses.

Fig. 6.

CFTR protein function is important for the S1611-stimulated DBS. The graph clearly shows that not the absence of CFTR, as in case of CFTR knockout (KO) mice, but CFTR function is important for S1611-stimulated DBS, since CFTR(inh)-172 significantly blocked the S1611-stimulated DBS. Results are means ± SE. *P < 0.05; ***P < 0.001, significant difference between 2 groups. #P < 0.05; ###P < 0.001, significant difference compared with basal response. The number of mice euthanized for the experiments is given in parenthesis.

Fig. 7.

Both Slc26a6 and Slc26a3 are important in electroneutral salt absorption in the small intestine. Fluid absorption in the jejunum of Slc26a6 (A) and Slc26a3 (B) KO and WT mice were measured and was significantly reduced in both the KO mice. The reduction in the Slc26a3 KO mouse was much more pronounced, indicating this might be responsible for the major portion of salt being absorbed. Results are means ± SE. *P < 0.05; **P < 0.01, significant difference between 2 groups. The number of mice euthanized for the experiments is given in parentheses.

Fig. 8.

Genetic deletion or pharmacological inhibition of CFTR did not alter the fluid absorptive capacity of small intestine. A: basal fluid absorption in the jejunum of CFTR KO mice was similar to hat in WT mice, indicating that the coupling of NHE3 with Slc26 members was not altered and electroneutral salt absorption was not compromised. Similar data were obtained when the experiment was performed in the duodenum (Fig. 5B). B: results of pharmacological inhibition of CFTR were similar to those for genetic deletion, indicating that both the inhibition of function of CFTR and total absence of CFTR did not alter the electroneutral salt absorption. Results are means ± SE. *P < 0.05; **P < 0.01, significant difference between 2 groups. The number of mice euthanized for the experiments is given in parentheses.

Fig. 9.

CFTR is important for stimulated-state bicarbonate secretion by various secretagogues. DBS was measured before and after various secretagogues were applied luminally (concentrations given in parentheses), and the net peak response was compared between CFTR KO and WT mice. The DBS response to all secretagogues used was significantly decreased in the case of KO mice. Results are means ± SE. ***P < 0.001, significant difference between 2 groups. The number of mice euthanized for the experiments are given in parenthesis.

Fig. 10.

Relative mRNA expression of CFTR, NHE3, Slc26a6, and Slc26a3 in the murine duodenum. The quantitative PCR data shown were acquired in procedures performed in WT mice against villin. We also used β-actin and cytokeratin 18 as control genes (see Supplementary Material, Supplemental Fig. 4). CFTR and NHE3 showed similar expression levels with any of the control genes.