Molecular mechanisms for intestinal HCO₃^-secretion and its regulation by guanylin in seawater-acclimated eels

Abstract

The intestine of marine teleosts secretes HCO 3 into the lumen and precipitates Ca 2+ and Mg 2+ in the imbibed seawater as carbonates to decrease luminal fluid osmolality and facilitate water absorption. However, reports on studies on the hormonal regulation of HCO 3 secretion are just emerging. Here, we showed that guanylin (GN) applied to the mucosal side of intestinal epithelia increased HCO 3 secretion in seawater-acclimated eels. The effect of GN on HCO 3 secretion was slower than that on the short-circuit current, and the time-course of the GN effect was similar to that of bumetanide. Mucosal bumetanide and serosal 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS) inhibited the GN effect, suggesting an involvement of apical Na + -K + -2Clcotransporter (NKCC2) and basolateral Cl -/HCO 3 exchanger (AE)/Na + -HCO 3 cotransporter (NBC) in the GN effect. However, mucosal DNDS and diphenylamine-2-carboxylic acid (DPC) failed to inhibit the GN effect, showing that apical AE and Clchannel are not involved. To identify molecular species of possible transporters involved in the GN effect, we performed RNA-seq analyses followed by quantitative real-time PCR after transfer of eels to seawater. Among the genes upregulated after seawater transfer, those of Slc26a3a, b (DRAa, b) and Slc26a6a, c (Pat-1a, c) on the apical membrane of the intestinal epithelial cells, and those of Sls4a4a (NBCe1a), Slc4a7 (NBCn1), Slc4a10a (NBCn2a) and Slc26a1 (Sat-1) on the basolateral membrane were candidate transporters involved in HCO 3 secretion. Judging from the slow effect of GN, we suggest that GN inhibits NKCC2b on the apical membrane and decreases cytosolic Cland Na + , which then activates apical DNDS-insensitive DRAa, b and basolateral DNDS-sensitive NBCela, n1, n2a to enhance transcellular HCO 3 flux across the intestinal epithelia of seawater-acclimated eels. Alper, S. L. and Sharma, A. K. (2013). The SLC26 gene family of anion transporters and channels. Mol. Aspects Med. 34, 494-515. Ando, M., and Subramanyam, M. V. V. (1990). Bicarbonate transport systems in the intestine of the seawater eel. J. Exp. Biol. 150, 381-394. Ando, M. and Takei, Y. (2015). Guanylin activates Clsecretion into the lumen of seawater eel intestine via apical Clchannel under simulated in vivo condition. Am. J. Physiol. 308, R400-R410. Ando, M., Wong, M. K. S. and Takei, Y. (2014). Mechanisms of guanylin action on water and ion absorption at different regions of seawater eel intestine. Am. J. Physiol. 307, R653-R663. Barmeyer, C., Ye, J. H., Sidani, S., Geibel, J., Binder, H. J. and Rajendran V. M. (2007).Characteristics of rat downregulated in adenoma (rDRA) expressed in HEK 293 cells. Pflü.Arch. 454, 441-450.