Martin Tresguerres

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 ( I_sc) in intestine mounted in Ussing chamber, bathed with the same saline solution on the external and internal sides of the epithelium. The I_scincreased 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 I_sc, and this [HCO₃⁻] was used in all subsequent experiments. The HCO₃⁻-stimulated I_scwas 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 I_scwas 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.

PH homeostasis is essential for life, yet it remains unclear how animals sense their systemic acid/base (A/B) status. Soluble adenylyl cyclase (sAC) is an evolutionary conserved signaling enzyme that produces the second messenger cAMP in response to bicarbonate ions (HCO₃⁻). We cloned the sAC ortholog from the dogfish, a shark that regulates blood A/B by absorbing and secreting protons (H⁺) and HCO₃⁻at its gills. Similar to mammalian sAC, dogfish soluble adenylyl cyclase (dfsAC) is activated by HCO₃⁻and can be inhibited by two structurally and mechanistically distinct small molecule inhibitors. dfsAC is expressed in the gill epithelium, where the subset of base-secreting cells resides. Injection of inhibitors into animals under alkaline stress confirmed that dfsAC is essential for maintaining systemic pH and HCO₃⁻levels in the whole organism. One of the downstream effects of dfsAC is to promote the insertion of vacuolar proton pumps into the basolateral membrane to absorb H⁺into the blood. sAC orthologs are present throughout metazoans, and mammalian sAC is expressed in A/B regulatory organs, suggesting that systemic A/B sensing via sAC is widespread in the animal kingdom.

Posterior isolated gills of Neohelice ( Chasmagnathus) granulatus were symmetrically perfused with hemolymph-like saline of varying [HCO₃⁻] and pH. Elevating [HCO₃⁻] in the saline from 2.5 to 12.5 mmol/l (pH 7.75 in both cases) induced a significant increase in the transepithelial potential difference ( V_te), a measure of ion transport. The elevation in [HCO₃⁻] also induced a switch from acid secretion (−43.7 ± 22.5 μequiv·kg⁻¹·h⁻¹) in controls to base secretion (84.7 ± 14.4 μequiv·kg⁻¹·h⁻¹). The HCO₃⁻-induced V_teincrease was inhibited by basolateral acetazolamide (200 μmol/l), amiloride (1 mmol/l), and ouabain (5 mmol/l) but not by bafilomycin (100 nmol/l). The V_teresponse to HCO₃⁻did not take place in Cl⁻-free conditions; however, it was unaffected by apical SITS (2 mmol/l) or DIDS (1 mmol/l). A decrease in pH from 7.75 to 7.45 pH units in the perfusate also induced a significant increase in V_te, which was matched by a net increase in acid secretion of 67.8 ± 18.4 μequiv kg⁻¹h⁻¹. This stimulation was sensitive to basolateral acetazolamide, bafilomycin, DIDS, and Na⁺-free conditions, but it still took place in Cl⁻-free saline. Therefore, the cellular response to low pH is different from the HCO₃⁻-stimulated response. We also report V-H⁺-ATPase- and Na⁺-K⁺-ATPase-like immunoreactivity in gill sections for the first time in this crab. Our results suggest that carbonic anhydrase (CA), basolateral Na⁺/H⁺exchangers and Na⁺-K⁺-ATPase and apical anion exchangers participate in the HCO₃⁻-stimulated response, while CA, apical V-H⁺-ATPase and basolateral HCO₃⁻-dependent cotransporters mediate the response to low pH.