The apical anion exchanger Slc26a6 promotes oxalate secretion by murine submandibular gland acinar cells

Abstract

The solute carrier family 26 (SLC26) gene family encodes at least 10 different anion exchangers. SLC26 member 6 (SLC26A6 or CFEX/PAT-1) and the cystic fibrosis transmembrane conductance regulator (CFTR) co-localize to the apical membrane of pancreatic duct cells, where they act in concert to drive HCO₃^- and fluid secretion. In contrast, in the small intestine, SLC26A6 serves as the major pathway for oxalate secretion. However, little is known about the function of Slc26a6 in murine salivary glands. Here, RNA sequencing-based transcriptional profiling and Western blots revealed that Slc26a6 is highly expressed in mouse submandibular and sublingual salivary glands. Slc26a6 localized to the apical membrane of salivary gland acinar cells with no detectable immunostaining in the ducts. CHO-K1 cells transfected with mouse Slc26a6 exchanged Cl^- for oxalate and HCO₃^-, whereas two other anion exchangers known to be expressed in salivary gland acinar cells, Slc4a4 and Slc4a9, mediated little, if any, Cl^-/oxalate exchange. Of note, both Cl^-/oxalate exchange and Cl^-/HCO₃^- exchange were significantly reduced in acinar cells isolated from the submandibular glands of Slc26a6^-/- mice. Oxalate secretion in submandibular saliva also decreased significantly in Slc26a6^-/- mice, but HCO₃^- secretion was unaffected. Taken together, our findings indicate that Slc26a6 is located at the apical membrane of salivary gland acinar cells, where it mediates Cl^-/oxalate exchange and plays a critical role in the secretion of oxalate into saliva.

Keywords: anion transport; epithelial cell; exchanger; glycoprotein; physiology.

Figure 1.

Slc26a6 mRNA expression in mouse salivary glands. A, Slc26a6 expression acquired by RNA-seq analysis for mouse PGs, SMGs, and SLGs are displayed as FPKM per 40 million mapped reads. Data from individual glands are displayed as circles (n = 6 for PG, SMG, and SLG). B, Slc26a6 mRNA expression levels were confirmed by qPCR and normalized to β-actin (Actb) (n = 6 for PG, SMG, and SLG). C, PCR band size and product amount for slc26a6 (149 bp) and Actb (147 bp) after PCR 27 cycles of cDNA amplification (100 ng). One-way ANOVA followed by Bonferroni's post hoc test was performed for statistical analysis; **, p < 0.01.

Figure 2.

Slc26a6 protein expression in mouse salivary glands. A, crude plasma membrane was isolated from PGs, SMGs, and SLGs. Each lane was loaded with 40 μg of protein and immunoblotted with mouse monoclonal antibodies (Ab) to Slc26a6 and β-actin. To more clearly visualize Slc26a6 protein expression in PGs, the blot was developed for 40 s (first lane). B, band intensities from A were quantified using ImageJ software and normalized to β-actin (n = 4). One-way ANOVA followed by Bonferroni's post hoc test was performed for statistical analysis; *, p < 0.002, **, p < 0.01. C, SMG (40 μg) and SLG (20 μg) plasma membrane proteins from Slc26a6^+/+ and Slc26a6^−/− mice and from Slc26a6-transfected CHO cells (20 μg) were treated without (−) or with (+) PNGaseF (PNG). The same membrane as in A was stripped and immunoblotted with an anti-β-actin antibody (n = 3).

Figure 3.

Slc26a6 localizes to the apical membranes of mouse salivary gland acinar cells. Shown is immunofluorescent staining of Slc26a6 (cyan), Cftr (green), and Nkcc1 (red). Nuclei were stained with 4′,6-diamidino-2-phenylindole (blue). A, Slc26a6 localized at the apical membrane of SMG acinar cells from Slc26a6^+/+ mice, with no overlap of apical duct Cftr (filled white arrow) or basolateral acinar Nkcc1 (filled red arrow) staining. B, high magnification image of Slc26a6 staining of the acinar cell apical membrane (open white arrow) from A. C, Slc26a6 was not detected in the SMGs of Slc26a6^−/− mice, whereas Nkcc1 staining was unchanged. D and E, Slc26a6 localized at the apical membrane of SLG acinar cells from Slc26a6^+/+ mice, with no Cftr or Nkcc1 overlap. F, Slc26a6 was not detected in the SMGs of Slc26a6^−/− mice, whereas Nkcc1 staining was unchanged. Representative images of three independent experiments are shown. Scale bars = 10 μm.

Figure 4.

Oxalate-dependent Cl⁻ uptake by mouse Slc26a6 in transfected CHO-K1 cells. CHO-K1 cells were transfected with plasmids containing either mouse Slc26a6, Ae2 (Slc4a2), or Ae4 (Slc4a9) cDNA, and the intracellular Cl⁻ concentration was measured using the Cl⁻-sensitive indicator SPQ. Intracellular Cl⁻ was initially depleting by exposure to a low Cl⁻ external solution, and then oxalate-dependent Cl⁻ uptake was induced by reintroduction of a high Cl⁻ solution. A, the high Cl⁻ solution induced little, if any, oxalate-dependent Cl⁻ uptake in Ae2-expressing (black circles, n = 26) or Ae4-expressing (gray circles, n = 18) CHO-K1 cells, whereas Slc26a6-transfected (gray squares, n = 26) CHO-K1 cells exhibited oxalate-dependent Cl⁻ uptake. Empty vector–transfected CHO cell did not induce Cl⁻ uptake (Empty, white circles, n = 15). B, summary of Cl⁻ uptake rates in the presence of oxalate. Data are presented as the mean ± S.E. of at least five cells per experiment from at least three different transfections. One-way ANOVA followed by Bonferroni's post hoc test was performed for statistical analysis; **, p < 0.01.

Figure 5.

Slc26a6 mediates Cl⁻/oxalate exchange in mouse SMG acinar cells. Acinar cells isolated from Slc26a6^+/+ and Slc26a6^−/− submandibular glands were loaded with SPQ to monitor Cl⁻ uptake. Cl⁻ depletion was induced by incubation in a low Cl⁻ solution in the absence (− Oxalate) or presence (+ Oxalate) of 25 mm oxalate, and then Cl⁻ uptake was stimulated by reintroduction of high extracellular Cl⁻ in the absence of oxalate. Experiments were performed in bicarbonate-free solutions containing ethoxyzolamide (30 μm B−/EZA) to eliminate Cl⁻/HCO₃⁻ exchange and T16Ainh-01 (10 μm A01) and bumetanide (Bumet, 80 μm) to inhibit Tmem16a and Nkcc1, respectively. A, oxalate-dependent Cl⁻ uptake was observed in Slc26a6^+/+ SMG acinar cells, but no Cl⁻ uptake was observed when oxalate was absent (+ Oxalate, open circles, n = 15; − Oxalate, closed circles, n = 15). B, Cl⁻/oxalate exchange activity was absent in SMG acinar cells from Slc26a6^−/− mice (+ Oxalate, open circles, n = 14; − Oxalate, closed circles, n = 16). C, summary of the Cl⁻ uptake experiments shown in A and B. Data are presented as the mean ± S.E. of cells isolated from at least four different mice per group. Statistical analysis was performed using unpaired t test; **, p < 0.01.

Figure 6.

Slc26a6 mediates oxalate secretion in response to β-adrenergic receptor stimulation. The ex vivo SMG was perfused in the presence (1 mm) or absence of oxalate and subsequently stimulated with the β-adrenergic receptor agonist IPR (1.0 μm) to induce saliva secretion. A, flow rate and flow amount (microliters) were comparable in Slc26a6^+/+ (n = 26) and Slc26a6^−/− (n = 23) mice. B, the oxalate concentration in saliva induced in the presence of oxalate was significantly less in Slc26a6^−/− mice, whereas the oxalate concentration in the oxalate-free solution was comparable for Slc26a6^+/+ (n = 17) and Slc26a6^−/− (n = 16) mice. C, net oxalate concentration = (oxalate concentration in oxalate-containing solution) minus (oxalate concentration in oxalate-free solution), subtracted from consecutive stimulations within an individual gland. Paired Student's t test was performed; **, p < 0.001. Results are given as the mean ± S.E.

Figure 7.

Acinar Cl⁻/HCO₃⁻ exchanger activity and isoproterenol-induced HCO₃⁻ secretion in SMGs from Slc26a6^−/− mice. A, intracellular alkalization was induced by Cl⁻/HCO₃⁻ exchange in SMG acinar cells loaded with the pH indicator BCECF by shifting from a high Cl⁻ to a low Cl⁻ bath solution in the presence of HCO₃⁻ (open circles, Slc26a6^+/+; closed circles, Slc26a6^−/−). B, summary of the alkalization rates from the data in A (B+) and in HCO₃⁻-free (B−) solutions. The alkalization rate in the presence of HCO₃⁻ was significantly reduced in Slc26a6^−/− SMG acinar cells (Slc26a6^+/+, B+ n = 17; Slc26a6^−/−, B+ n = 9), whereas alkalization was essentially eliminated under HCO₃⁻-free conditions (Slc26a6^+/+, B− n = 16; Slc26a6^−/−, B− n = 11). C, an ex vivo SMG was perfused with a 25 mm HCO₃⁻- containing solution and stimulated with the β-adrenergic receptor agonist IPR (1.0 μm). Flow rate and flow amount (microliters) were comparable in Slc26a6^+/+ (n = 22) and Slc26a6^−/− (n = 23) mice. D, bicarbonate concentration in saliva from Slc26a6^+/+ and Slc26a6^−/− mice were comparable (Slc26a6^+/+, n = 14; Slc26a6^−/−, n = 13). Unpaired Student's t test was performed; *, p < 0.05. Results are given as mean ± S.E.