In vertebrate salt-secreting epithelia, Na + moves passively down an electrochemical gradient via a paracellular pathway. We assessed how this pathway is modified to allow Na + secretion in hypersaline environments. Mummichogs (Fundulus heteroclitus) acclimated to hypersaline [2× seawater (2SW), 64‰] for 30 days developed invasive projections of accessory cells with an increased area of tight junctions, detected by punctate distribution of CFTR (cystic fibrosis transmembrane conductance regulator) immunofluorescence and transmission electron miscroscopy of the opercular epithelia, which form a gill-like tissue rich in ionocytes. Distribution of CFTR was not explained by membrane raft organization, because chlorpromazine (50 μmol l ) did not affect opercular epithelia electrophysiology. Isolated opercular epithelia bathed in SW on the mucosal side had a transepithelial potential (V t ) of +40.1±0.9 mV (N=24), sufficient for passive Na + secretion (Nernst equilibrium voltage≡E Na =+24.11 mV). Opercular epithelia from fish acclimated to 2SW and bathed in 2SW had higher V t of +45.1±1.2 mV (N=24), sufficient for passive Na + secretion (E Na =+40.74 mV), but with diminished net driving force. Bumetanide block of Cl − secretion reduced V t by 45% and 29% in SW and 2SW, respectively, a decrease in the driving force for Na + extrusion. Estimates of shunt conductance from epithelial conductance (G t ) versus short-circuit current (I sc ) plots (extrapolation to zero I sc ) suggested a reduction in total epithelial shunt conductance in 2SW-acclimated fish. In contrast, the morphological elaboration of tight junctions, leading to an increase in accessory-cell-ionocyte contact points, suggests an increase in local paracellular conductance, compensating for the diminished net driving force for Na + and allowing salt secretion, even in extreme salinities.
Ionocytes of euryhaline teleost fish secrete NaCl, under regulation by serine and threonine kinases, including with-no-lysine kinase (WNK1) and p38 mitogen-activated protein kinase (MAPK). Mummichogs (Fundulus heteroclitus L.) were acclimated to freshwater (FW), full strength seawater (SW) and hypersaline conditions (2SW). Immunocytochemistry of ionocytes in opercular epithelia of fish acclimated to SW and 2SW revealed that WNK1-anti-pT58 phosphoantibody localized strongly to accessory cells and was present in the cytosol of ionocytes, close to cystic fibrosis transmembrane conductance regulator (CFTR) in the apical membrane and the sodium potassium 2 chloride cotransporter (NKCC) in the basolateral membrane. In FW acclimated fish, WNK1 localized to a sub-apical zone, did not colocalize with apical membrane-located sodium chloride cotransporter (NCC), and typically was present in one cell of paired ionocytes and in some single ionocytes. Forskolin treatment (10 μM, 30 min) increased WNK1 immunofluorescence in SW ionocytes only, while hypertonicity had little effect, compared to controls. Anti-p38-MAPK antibody localized to the cytosolic compartment. The distribution of WNK1 and p38MAPK is consistent with a proximal position in regulatory cascades, rather than directly affecting transporters. The strong staining of accessory cells by WNK1 phosphoantibody infers an osmoregulatory function for WNK.
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