The Role of Uroguanylin in Regulation of Ion Transport in Salivary Glands

Abstract

Objectives: Guanylin peptides are considered to be the only intrinsic regulators of salivary glands secretion. Therefore, the aim of this study was to determine the effects of systemic uroguanylin (UGN) of the salivary flow and ion composition. Besides, the objective was to investigate whether those effects include activation of guanylate cyclase C (GC-C).

Material and methods: This study was conducted on 7 months old C57Bl6NCrl (wild type, WT) and GC-C knockout (KO) mice. Salivary flow rate and ion composition were determined after pilocarpine stimulation with UGN (30 µg/animal) or saline i.p. application. The expression of mRNA for AQPs, NHEs, NBCn1, Slc26a3/a6 and CFTR were determined by qPCR in submandibular salivary glands.

Results: When applied i.p., UGN decreased the pilocarpine stimulated saliva flow rate and increased the concentration of Na⁺, H⁺ and Cl^-. In GC-C KO mice, UGN showed no effect on saliva flow rate, while the concentrations of Na⁺, H⁺ and Cl^- are the same in GC-C KO littermates when compared to WT mice. UGN increased expression of Slc26a6 while in GC-C KO mice Slc26a6 had a higher expression when compared to WT mice, suggesting involvement of GC-C independent signalling pathway for UGN. The difference in Slc26a6 in GC-C KO mice is not unique for salivary glands because it was also found in duodenum and kidney cortex.

Conclusions: The effects of UGN via basolateral membrane of salivary glands cells have not been considered up to date. In our study, UGN, when applied i.p., decreased salivary flow rate, pH, and changed the composition of other ions. Therefore, plasma UGN, an hour after a meal, could have physiological and pathological importance (development of cavities, inflammations or demineralizations), and the inhibition of systemic UGN effects could be considered a new approach in treatment of those conditions.

Keywords: Enterotoxin Receptors; GC-C Independent Signaling Pathway; MeSH Terms: Submandibular Gland; Pilocarpine; Saliva Flow Rate; Salivation; Stimulated Saliva Production; pH; qPCR.

Figure 1

Uroguanylin decreased pilocarpine stimulated saliva flow rate and pH via different signalling pathways. A: Effects of uroguanylin (UGN) on saliva production was not present in guanylate cyclase C (GC-C) knockout mice missing GC-C receptor (GC-C KO). B: UGN decreased pH, but this effect is still present in GC-C KO mice. The results are presented as mean ± SEM, n = 6-8. *p < 0.05 statistically significant when compared to WT control; **p < 0.05 statistically significant when compared to GC-C WT which are siblings (littermates) of GC-C KO mice but still have GC-C receptor. WT – wild type mice

Figure 2

Uroguanylin increased Na⁺ and Cl^- concentration via GC-C independent signalling pathway. Uroguanylin (UGN) increased concentration of Na⁺ and Cl^- and that effect is still present in GC-C KO mice. UGN did not change K⁺ concentration. The results are presented as mean ± SEM, n = 6-8. *p < 0.05 statistically significant when compared to WT control; WT – wild type mice

Figure 4

Expression of members of Slc26 family increased in submandibular salivary gland, duodenum and kidney cortex of animals missing GC-C. *p < 0.05 statistically significant when compared to GC-C WT mice (n = 6-10). The results are presented as mean ± SEM. GC-C KO - guanylate cyclase C (GC-C) knockout mice missing GC-C receptor

Figure 3

Uroguanylin increased mRNA expression of Slc26a6 in submandibular salivary glands via GC-C independent signalling pathway. A: Effects of uroguanylin (UGN) on expression of channels for water (AQPs), sodium-hydrogen exchanger isoform 1 (NHE1) and 3 (NHE3), sodium-bicarbonate cotransporter (NBCn1), solute carrier family 26 member 6 (Slc26a6), and cystic fibrosis transmembrane conductance regulator (CFTR) are shown. *p < 0.05 statistically significant when compared to WT Control animals, n = 7-9. B: Missing GC-C in submandibular glands of GC-C KO mice lead to an increase in NHE3 and Slc26a6 expression (*p < 0.05 statistically significant when compared to GC-C WT mice, n = 7-10). The results are presented as mean ± SEM. GC-C KO - guanylate cyclase C (GC-C) knockout mice missing GC-C receptor, 26A6 – Slc26a6, WT – wild type mice