Putative anion transporter-1 (Pat-1, Slc26a6) contributes to intracellular pH regulation during H+-dipeptide transport in duodenal villous epithelium

Abstract

The majority of dietary amino acids are absorbed via the H(+)-di-/tripeptide transporter Pept1 of the small intestine. Proton influx via Pept1 requires maintenance of intracellular pH (pH(i)) to sustain the driving force for peptide absorption. The apical membrane Na(+)/H(+) exchanger Nhe3 plays a major role in minimizing epithelial acidification during H(+)-di-/tripeptide absorption. However, the contributions of HCO(3)(-)-dependent transporters to this process have not been elucidated. In this study, we investigate the role of putative anion transporter-1 (Pat-1), an apical membrane anion exchanger, in epithelial pH(i) regulation during H(+)-peptide absorption. Using wild-type (WT) and Pat-1(-) mice, Ussing chambers were employed to measure the short-circuit current (I(sc)) associated with Pept1-mediated glycyl-sarcosine (Gly-Sar) absorption. Microfluorometry was used to measure pH(i) and Cl(-)/HCO(3)(-) exchange in the upper villous epithelium. In CO(2)/HCO(3)(-)-buffered Ringers, WT small intestine showed significant Gly-Sar-induced I(sc) and efficient pH(i) regulation during pharmacological inhibition of Nhe3 activity. In contrast, epithelial acidification and reduced I(sc) response to Gly-Sar exposure occurred during pharmacological inhibition of Cl(-)/HCO(3)(-) exchange and in the Pat-1(-) intestine. Pat-1 interacts with carbonic anhydrase II (CAII), and studies using CAII(-) intestine or the pharmacological inhibitor methazolamide on WT intestine resulted in increased epithelial acidification during Gly-Sar exposure. Increased epithelial acidification during Gly-Sar exposure also occurred in WT intestine during inhibition of luminal extracellular CA activity. Measurement of Cl(-)/HCO(3)(-) exchange in the presence of Gly-Sar revealed an increased rate of Cl(-)(OUT)/HCO(3)(-)(IN) exchange that was both Pat-1 dependent and CA dependent. In conclusion, Pat-1 Cl(-)/HCO(3)(-) exchange contributes to pH(i) regulation in the villous epithelium during H(+)-dipeptide absorption, possibly by providing a HCO(3)(-) import pathway.

Fig. 1.

Glycyl-sarcosine (Gly-Sar)-induced short-circuit current (I_sc) response and intracellular pH (pH_i) acidification in wild-type (WT) murine proximal intestine. A: I_sc response to luminal addition of 20 mM Gly-Sar in the absence (control) or presence of EIPA (EIPA, 100 μM, luminal). *Significantly different from vehicle control (n = 5–8). B: representative trace of villous epithelial pH_i during luminal addition of 20 mM Gly-Sar in the absence of CO₂/HCO₃⁻ in the superfusate (TES-buffered). C: representative trace of villous epithelial pH_i during luminal addition of 20 mM Gly-Sar in the presence of CO₂/HCO₃⁻ buffering in the superfusate. D: rates of Gly-Sar-induced intracellular acidification in WT upper villous epithelium in the absence of CO₂/HCO₃⁻ (without CO₂/HCO₃⁻), in the presence of CO₂/HCO₃⁻ (with CO₂/HCO₃⁻), and in the presence of CO₂/HCO₃⁻ during luminal treatment with 100 μM EIPA (with CO₂/HCO₃⁻ + EIPA) (n = 3–6). The mean pH_i before Gly-Sar addition for each condition was pH_i (without CO₂/HCO₃⁻) = 7.78 ± 0.11, pH_i (with CO₂/HCO₃⁻) = 7.68 ± 0.09, and pH_i (with CO₂/HCO₃⁻ + EIPA) = 7.59 ± 0.19. ^a,bMeans with the same letters are not significantly different.

Fig. 2.

Gly-Sar-induced I_sc response and pH_i acidification in WT murine proximal intestine during inhibition of apical membrane Cl⁻/HCO₃⁻ exchange. A: I_sc response to luminal treatment with 20 mM Gly-Sar for WT intestine in the absence (control) or presence of niflumic acid (NFA, 100 μM, luminal). *Significantly different from vehicle control (n = 4). B: summary of acidification rates for WT duodenal villous epithelial cells during luminal treatment with 20 mM Gly-Sar in the upper villous epithelium in Krebs bicarbonate Ringers (KBR) solution (control), in the presence of NFA, and in the absence of luminal Cl⁻ (Cl⁻ Free). The mean pH_i before Gly-Sar addition for each condition was Control pH_i = 7.68 ± 0.09, NFA pH_i = 7.66 ± 0.08, and Cl⁻-free pH_i = 7.75 ± 0.16. ^a,b,cMeans with the same letters are not significantly different.

Fig. 3.

Gly-Sar-induced I_sc response and pH_i acidification in the proximal small intestine of WT and putative anion transporter-1 (Pat-1)(−) mice. A: I_sc response to luminal addition of 20 mM Gly-Sar in WT and Pat-1(−) intestines in physiological KBR (left) and during luminal treatment with 100 μM EIPA (right). Changes in the I_sc (ΔI_sc = basal I_sc − Gly-Sar I_sc) at each time point after Gly-Sar addition are shown. *Significantly different from WT (n = 13–14). B: summary of acidification and alkalinization rates for upper villous epithelium of WT and Pat-1(−) duodena in physiological KBR solution during luminal addition and removal of 20 mM Gly-Sar, respectively. The mean pH_i before Gly-Sar addition was WT pH_i = 7.68 ± 0.07 and Pat-1(−) pH_i = 7.58 ± 0.11 (n = 3). *Significantly different from WT. C: steady-state pH_i under control conditions and during luminal exposure to 20 mM Gly-Sar in villous epithelium of WT and Pat-1(−) duodena superfused in physiological KBR solution (n = 4). *Significantly different from control.

Fig. 4.

Gly-Sar-induced pH_i acidification in the proximal small intestine during inhibition of carbonic anhydrases (CAs) and in the CAII(−) knockout mouse. A: summary of acidification rates for WT villous epithelium during luminal addition of 20 mM Gly-Sar in the absence (control) or presence of methazolamide (Methaz, 100 μM, bilateral addition). The mean pH_i before Gly-Sar addition was control pH_i = 7.67 ± 0.27 and Methaz pH_i = 7.63 ± 0.06. *Significantly different from vehicle control (n = 3). B: summary of acidification rates for upper villous epithelial cells during luminal addition of 20 mM Gly-Sar in WT and CAII(−) duodena. The mean pH_i before Gly-Sar addition was WT pH_i = 7.74 ± 0.10 and CAII(−) pH_i = 7.61 ± 0.08. *Significantly different from WT (n = 4). C: summary of acidification rates for WT villous epithelium during luminal exposure to 20 mM Gly-Sar (control) followed by treatment with the extracellular CA inhibitor, sulphonamide compound 1-[4-aminosulphonyl] phenyl-2,4,6-trimethylpyridinium perchlorate (6a) (30 μM, luminal addition for 5 min). The mean pH_i before Gly-Sar addition was 7.35 ± 0.06. *Significantly different from control (n = 3).

Fig. 5.

Comparison of Cl⁻/HCO₃⁻ exchange activity in upper villous epithelium of WT and Pat-1(−) duodena during luminal treatment with 20 mM Gly-Sar. A and B: rates of Cl⁻_OUT/HCO₃⁻_IN and Cl⁻_IN/HCO₃⁻_OUT exchange of WT upper villous epithelium induced by luminal Cl⁻ removal and replacement, respectively, during vehicle treatment (control), in the presence of Gly-Sar (20 mM, luminal), and in the presence of Gly-Sar during methazolamide (100 μM, bilateral addition) treatment. Duodenal preparations were exposed to 20 mM Gly-Sar for 20 min either in the presence or absence of methazolamide before measurement of Cl⁻/HCO₃⁻ exchange rates. The mean pH_i before measurement of Cl⁻/HCO₃⁻ exchange rates for each condition were control pH_i = 7.62 ± 0.18, Gly-Sar pH_i = 7.67 ± 0.27, and Gly-Sar + Methaz pH_i = 7.63 ± 0.06. ^a,bMeans with the same letters are not significantly different (n = 5–6). C and D: rates of Cl⁻_OUT/HCO₃⁻_IN and Cl⁻_IN/HCO₃⁻_OUT exchange of upper villous epithelium induced by luminal Cl⁻ removal and replacement, respectively, in Pat-1(−) duodenum under control conditions in KBR solution (open bar) and after 20-min exposure to 20 mM Gly-Sar (solid bar). The mean pH_i before measurement of Cl⁻/HCO₃⁻ exchange rates were Pat-1(−) pH_i 7.57 ± 0.06 and Pat-1(−) + Gly-Sar pH_i = 7.44 ± 0.12 (n = 3–4).

Fig. 6.

Cell model depicting the contributions of Nhe3, Pat-1, and CAs to pH_i regulation during H⁺-dipeptide absorption via Pept1.