WNK1 and p38-MAPK distribution in ionocytes and accessory cells of euryhaline teleost fish implies ionoregulatory function

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 sodium, potassium, 2 chloride cotransporter (NKCC) in basolateral membrane. In FW acclimated fish, WNK1 localized to a sub-apical zone not colocalized with apical membrane-located sodium chloride cotransporter (NCC) and typically was present in one cell of paired ionocytes and was present in some single ionocytes. Forskolin treatment (10 µM, 30 min) increased immunofluorescence for WNK1 of SW ionocytes only, while hypertonicity had little effect, compared to SW 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 a osmoregulatory function for WNK.