%0 Journal Article %A Yoshio Takei %A Marty K.S. Wong %A Masaaki Ando %T Molecular mechanisms for intestinal HCO3- secretion and its regulation by guanylin in seawater-acclimated eels %D 2019 %R 10.1101/580761 %J bioRxiv %P 580761 %X The intestine of marine teleosts secretes HCO3- into the lumen and precipitates Ca2+ and Mg2+ in the imbibed seawater as carbonates to decrease luminal fluid osmolality and facilitate water absorption. However, reports on studies on the hormonal regulation of HCO3- secretion are just emerging. Here, we showed that guanylin (GN) applied to the mucosal side of intestinal epithelia increased HCO3- secretion in seawater-acclimated eels. The effect of GN on HCO3- 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+-2Cl- cotransporter (NKCC2) and basolateral Cl-/HCO3- exchanger (AE)/Na+-HCO3- 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 Cl- channel 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 HCO3- secretion. Judging from the slow effect of GN, we suggest that GN inhibits NKCC2b on the apical membrane and decreases cytosolic Cl- and Na+, which then activates apical DNDS-insensitive DRAa, b and basolateral DNDS-sensitive NBCela, n1, n2a to enhance transcellular HCO3- flux across the intestinal epithelia of seawater-acclimated eels.AEanion exchanger;CAcarbonic anhydrase;CFTRcystic fibrosis transmembrane conductance regulator;DIDS4,4’-diisothiocyano-2,2’-disulfonic acid;DMSOdimethyl sulfoxide;DNDS4,4’-dinitrostilbene-2,2’-disulfonic acid;DPCdiphenylamine-2-carboxylic acidDRAdown-regulated in adenoma;FWfresh water;GNguanylin;Iscshort-circuit current;NBCNa+- HCO3- cotransporter;NCCNa+-Cl- cotransporter;NKANa+/K+-ATPase;NKCCNa+-K+-2Cl- cotransporter;Pat-1Putative anion transporter-1;PDpotential difference;Sat-1Sulfate transporter-1;SlcSolute Carrier;SWseawater %U https://www.biorxiv.org/content/biorxiv/early/2019/03/18/580761.full.pdf