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 gil l 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 kidn ey tissue. Rubidium uptake was linear for at least 30 min and was significa ntly inhibited by 1 mmol 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 l(-1) dibutyryl-cyclic AMP, 1 mmol l(- 1) theophylline, 10 mu mol l(-1) forskolin and 10 mu mol l(-1) isoprotereno l) and kidney (10Clmoll-l forskolin) tissue from freshwater-acclimated fish . In a separate series of experiments, ATP hydrolase activity was assayed i n a permeabilised gill membrane preparation after incubation of tissue bloc ks with 10 mu mol l(-1) forskolin, Forskolin elevated gill cyclic AMP level s 40-fold, inhibited maximal enzymatic Na+/K+-ATPase activity (Vmax) in gil l tissue from both freshwater- and seawater-acclimated fish and reduced the apparent K+ affinity in the gills of seawater-acclimated fish, demonstrati ng 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+d-ATPase activity, indicating that forskolin-mediated modulation was stable for the duration of assay. W e suggest that cyclic-AMP-mediated phosphorylation through protein kinases may underlie the rapid modulation of Na+/K+-ATPase activity in the osmoregu latory tissues of euryhaline teleosts.