Rapid modulation of Na+/K+-ATPase activity in osmoregulatory tissues of a salmonid fish

The effects of cyclic AMP on Na+/K+-ATPase activity were studied in the gill and kidney of the euryhaline brown trout Salmo trutta using two different experimental approaches. In the first series of experiments, in situ Na+/K+-ATPase activity was analyzed by measuring the ouabain-sensitive uptake of non-radioactive rubidium (Rb+) into gill cells and blocks of gill and kidney tissue. Rubidium uptake was linear for at least 30 min and was significantly inhibited by 1 mmol x l(-1) ouabain. Several agents presumed to increase the intracellular cyclic AMP concentration inhibited ouabain-sensitive Rb+ uptake in both gill (0.5 and 2 mmol x l(-1) dibutyryl-cyclic AMP, 1 mmol x l(-1) theophylline, 10 micromol x l(-1) forskolin and 10 micromol x l(-1 )isoproterenol) and kidney (10 micromol x l(-1) forskolin) tissue from freshwater-acclimated fish. In a separate series of experiments, ATP hydrolase activity was assayed in a permeabilised gill membrane preparation after incubation of tissue blocks with 10 micromol x l(-1 )forskolin. Forskolin elevated gill cyclic AMP levels 40-fold, inhibited maximal enzymatic Na+/K+-ATPase activity (Vmax) in gill tissue from both freshwater- and seawater-acclimated fish and reduced the apparent K+ affinity in the gills of seawater-acclimated fish, demonstrating that the effects are mediated through modifications of the enzyme itself. The protein phosphatase inhibitors okadaic acid and cyclosporin A did not affect forskolin-induced inhibition of Na+/K+-ATPase activity, indicating that forskolin-mediated modulation was stable for the duration of assay. We suggest that cyclic-AMP-mediated phosphorylation through protein kinases may underlie the rapid modulation of Na+/K+-ATPase activity in the osmoregulatory tissues of euryhaline teleosts.