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

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, the hormonal regulation of HCO₃^- secretion is largely unknown. Here, mucosally added guanylin (GN) increased HCO₃^- secretion, measured by pH-stat, across isolated seawater-acclimated eel intestine bathed in saline at pH 7.4 (5% CO₂). 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. As mucosal DNDS failed to inhibit the GN effect, apical DNDS-sensitive AE may not be involved. To identify molecular species of 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, AE genes (draa, b, and pat1a, c) on the apical membrane, and NBC genes (nbce1a, n1, n2a) and an AE gene (sat-1) on the basolateral membrane were candidates 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 DRAs and basolateral DNDS-sensitive NBCs to enhance transcellular HCO₃^- flux across the intestinal epithelia of seawater-acclimated eels.