Bicarbonate exchangers SLC26A3 and SLC26A6 are localized at the apical membrane of porcine vas deferens epithelium

Abstract

The goal of this study was to test for expression of HCO3 (-) exchangers SLC26A3 and SLC26A6 in primary cultures of porcine vas deferens epithelial cells (1°PVD) and native porcine vas deferens. Quantitative RT-PCR revealed that mRNA coding for SLC26A6 was six times more abundant than mRNA coding for SLC26A3 in 1°PVD cells. Western blot analyses combined with surface biotinylation of 1°PVD demonstrated SLC26A3 and SLC26A6 immunoreactivities in whole-cell lysates and apical surfaces of monolayers. Laser scanning confocal microscopy (LSCM) of the 1°PVD cell monolayers demonstrated that SLC26A3 immunoreactivity was primarily in the apical region but present throughout the basal-apical cellular axis, whereas SLC26A6 immunoreactivity was present in the apical region and sometimes accumulated in the nuclear region. LSCM also demonstrated SLC26A3 and SLC26A6 immunoreactivities present along the entire apical lining of the native porcine vas deferens epithelium and in basal cells. The patterns and apparent abundance of SLC26A3 and SLC26A6 immunoreactivities in the proximal vas deferens were not different from the corresponding immunoreactivities in the distal region. There is no evidence of preferential expression of SLC26A3 or SLC26A6 in any portion of the vas deferens, as has been proposed for epithelia that secrete HCO3 (-) in other duct systems. Thus, vas deferens epithelia express transporters throughout the duct that can contribute to rapid alkalinization of the luminal contents as it has been demonstrated in vivo.

Keywords: Epithelia; ion transport; vas deferens.

Figure 1

Primary cultures of porcine vas deferens epithelial (1°PVD) cells express HCO₃⁻ transporters. (A) Number of mRNA copies coding for SLC26A3 and SLC26A6 in RNA isolated from 1°PVD cells. (B) Gel images demonstrate a single amplicon with expected mobility following single target qRT-PCR conducted with primers for SLC26A3 (424 bp) and SLC26A6 (494 bp). Lane 1, DNA ladder; lanes 2–6, RT-PCR products derived from five RNA samples that were isolated from 1°PVD monolayers (five different animals). (C) Western blot analyses reveal SLC26A3 and SLC26A6 immunoreactivities in whole-cell lysates (WCL) derived from 1°PVDs. Blots were probed with 0.5 μg/mL of anti-SLC26A3 antibody or anti-SLC26A6 antibodies (WCL), or probed with antibodies preadsorbed to immunizing peptide. Arrows indicate major bands detected. Results are representative of six 1°PVD monolayers (six different animals).

Figure 2

SLC26A3 (A) and SLC26A6 (B) immunoreactivities (green) are observed in primary cultures of 1°PVD cells. Images are from the optical slice with greatest anti-occludin immunoreactivity (red). Top and right side images represent orthogonal projections in the X-Z and Y-Z dimensions, respectively. Orthogonal images show apical location of occludin immunoreactivity. SLC26A3 and SLC26A6 immunoreactivities are greatest at the apical aspect, but diffuse labeling is present throughout the cells. No signal was observed when primary antibodies were preadsorbed. DAPI (blue) was used to demonstrate the nuclei location. White scale bar represents 10 μm. (C) Distribution curves of the mean normalized voxels number (mean ± SD) for occludin, SLC26A3 and SLC26A6 immunoreactivities and for DAPI. Four monolayers from four boars were studied. Inset demonstrates the anti-SLC26A6 labeling for each of the four monolayers demonstrating two distinct labeling profiles.

Figure 3

Bicarbonate exchangers SLC26A3 and SLC26A6 are expressed at the apical membrane of cells lining porcine vas deferens. (A) Images from typical transverse sections of intact snap-frozen vas deferens. A lumen (L) is lined by epithelia. Cellular nuclei are labeled in blue (DAPI). Scale bar represents 100 μm. (B and C) Typical patterns of SLC26A3 and SLC26A6 immunoreactivities (green) are shown. Intense SLC26A3 and SLC26A6 immunoreactivities were located at or near the apical aspect of principal cells (dashed-line box frames a few principal cells) throughout proximal and distal segments of porcine vas deferens. SLC26A3 immunoreactivity was intense in sperm heads (circled by ovals). SLC26A6, and to a lesser extent SLC26A3, immunoreactivity was also observed in the area of basal cells (solid-line box frames a portion of this area). No signal was detected when primary antibodies were preadsorbed by their respective immunizing peptides. (D) No signal was detected when primary antibodies were omitted. Scale bar represents 50 μm for panels B, C, and D. Proximal and distal segments from seven animals were studied.

Figure 4

HCO₃⁻ exchangers SLC26A3 and SLC26A6 are expressed in the apical membrane of 1°PVD cells as indicated by surface biotinylation. WCL, whole-cell lysate; Vehicle, represents WCL from monolayers not exposed to biotin, but subjected to the same protein isolation protocol; Apical or Basolateral indicates the surface exposed to biotin; Flow through represents the first eluate collected from the avidin column and represents the intracellular fraction of protein not accessible to extracellular biotinylation. Positions of standard mobility markers are shown on the right. Images are representative of 4 (A) and 3 (B) boars.