Modulation of NaCl absorption by [HCO3−] in the marine teleost intestine is mediated by soluble adenylyl cyclase

Abstract

Intestinal HCO3−secretion and NaCl absorption are essential for counteracting dehydration in marine teleost fish. We investigated how these two processes are coordinated in toadfish. HCO3−stimulated a luminal positive short-circuit current ( Isc) in intestine mounted in Ussing chamber, bathed with the same saline solution on the external and internal sides of the epithelium. The Iscincreased proportionally to the [HCO3−] in the bath up to 80 mM NaHCO3, and it did not occur when NaHCO3was replaced with Na+-gluconate or with NaHCO3in Cl−-free saline. HCO3−(20 mM) induced a ∼2.5-fold stimulation of Isc, and this [HCO3−] was used in all subsequent experiments. The HCO3−-stimulated Iscwas prevented or abolished by apical application of 10 μM bumetanide (a specific inhibitor of NKCC) and by 30 μM 4-catechol estrogen [CE; an inhibitor of soluble adenylyl cyclase (sAC)]. The inhibitory effects of bumetanide and CE were not additive. The HCO3−-stimulated Iscwas prevented by apical bafilomycin (1 μM) and etoxolamide (1 mM), indicating involvement of V-H+-ATPase and carbonic anhydrases, respectively. Immunohistochemistry and Western blot analysis confirmed the presence of an NKCC2-like protein in the apical membrane and subapical area of epithelial intestinal cells, of Na+/K+-ATPase in basolateral membranes, and of an sAC-like protein in the cytoplasm. We propose that sAC regulates NKCC activity in response to luminal HCO3−, and that V-H+-ATPase and intracellular carbonic anhydrase are essential for transducing luminal HCO3−into the cell by CO2/HCO3−hydration/dehydration. This mechanism putatively coordinates HCO3−secretion with NaCl and water absorption in toadfish intestine.