Immunolocalization of electroneutral Na⁺-HCO 3 − cotransporters in human and rat salivary glands

Abstract

Patterns of salivary HCO secretion vary widely among species and among individual glands. In particular, virtually nothing is known about the molecular identity of the HCO transporters involved in human salivary secretion. We have therefore examined the distribution of several known members of the Na(+)-HCO cotransporter (NBC) family in the parotid and submandibular glands. By use of a combination of RT-PCR and immunoblotting analyses, the electroneutral cotransporters NBC3 and NBCn1 mRNA and protein expression were detected in both human and rat tissues. Immunohistochemistry demonstrated that NBC3 was present at the apical membranes of acinar and duct cells in both human and rat parotid and submandibular glands. NBCn1 was strongly expressed at the basolateral membrane of striated duct cells but not in the acinar cells in the human salivary glands, whereas little or no NBCn1 labeling was observed in the rat salivary glands. The presence of NBCn1 at the basolateral membrane of human striated duct cells suggests that it may contribute to ductal HCO secretion. In contrast, the expression of NBC3 at the apical membranes of acinar and duct cells in both human and rat salivary glands indicates a possible role of this isoform in HCO salvage under resting conditions.

Electrogenic NBCe1 (SLC4A4), but not electroneutral NBCn1 (SLC4A7), cotransporter undergoes cholinergic-stimulated endocytosis in salivary ParC5 cells

“…In contrast to our detection of electroneutral cotransporter at the BLM in ParC5 cells, Gresz et al (21) did not find any NBCn1 in rat parotid acini. Gresz et al (21) used an indirect immunoperoxidase labeling of dewaxed and rehydrated paraffin tissue sections, whereas here we used confocal immunofluorescence microscopy in fixed cultured cells.…”

“…Gresz et al (21) used an indirect immunoperoxidase labeling of dewaxed and rehydrated paraffin tissue sections, whereas here we used confocal immunofluorescence microscopy in fixed cultured cells. We also support our finding with Western blot analysis of the biotinylated NBCn1 protein extracted from ParC5 cells (Fig.…”

An anti-NH2-terminal antibody localizes NBCn1 to heart endothelia and skeletal and vascular smooth muscle cells

“…NBCn1 staining was primarily found in vascular and ductal structures, which comprise a small fraction of the tissue, and these structures would not be well represented in the loaded proteins in immunoblotting. Nevertheless, previous studies have detected NBCn1 in the rat parotid and submandibular glands (9). In that study, the presence of NBCn1 RNA in these tissues was confirmed by immunoblotting, but immunohistochemical analysis only showed weak labeling of submandibular gland and no labeling of parotid gland.…”

“…The NBCn1 has been localized to the basolateral membrane of other renal tubules as type A intercalated cells of collecting ducts and terminal inner medullary collecting ducts (15,20). In other epithelia, NBCn1 is also a basolateral protein as in the duodenal mucosa, choroid plexus, and salivary glands (9,14,16). Recently, NBCn1 was also found in the rat hippocampal neurons (7).…”

Regulation of the human NBC3 Na+/HCO3−cotransporter by carbonic anhydrase II and PKA

“…NBC3, originally cloned from human skeletal muscle, was also localized by multiple-tissue Northern blot analysis of the heart (23). Subsequently, NBC3 has been identified at the apical surface of several epithelial cell types including renal outer medullary collecting duct type A intercalated cells and duct and acinar cells of parotid and submandibular glands (10,17,24). A recent report evaluated the contribution of Na ϩ -dependent transepithelial HCO 3 Ϫ flux in outer medullary collecting duct preparations and found that this transport mechanism provided only a minor contribution to the total transepithelial HCO 3 Ϫ flux (43).…”