Synthesis of gill Na+-K+-ATPase in Atlantic salmon smolts: differences in alpha-mRNA and alpha-protein levels

Several parameters were analyzed to determine the mechanisms responsible fo r the enhancement of the gill Na+-K+-ATPase activity of Atlantic salmon smo lts. A major alpha-subunit transcript of 3.7 kb was revealed by Northern bl ot in both parr and smolt gills when hybridized with two distinct cDNA prob es. The alpha-mRNA abundance demonstrated an increase to maximal levels in smolts at an early stage of the parr-smelt transformation. This was followe d by a gradual rise in alpha-protein levels, revealed by Western blots with specific antibodies and by an increase in gill Na+-K+-ATPase hydrolytic ac tivity, both only reaching maximum levels a month later, at the peak of the transformation process. Parr fish experienced a decrease in alpha-mRNA abu ndance and had basal levels of alpha-protein and enzyme activity. Measureme nt of the binding of [H-3] ouabain to Na+-K+-ATPase was characterized in sm olts and parr gill membranes showing more than a twofold elevation in smolt s and was of high affinity in both groups (dissociation constant = 20-23 nM ). Modulation of the enzyme due to increased salinity was also observed in seawater-transferred smolts, as demonstrated by an increase in alpha-mRNA l evels after 24 h with a rise in Na+-K+-ATPase activity occurring only after 11 days. No qualitative change in alpha-expression was revealed at either the mRNA or protein level. Immunological identification of the alpha-protei n was performed with polyclonal antibodies directed against the rat alpha-s pecific isoforms, revealing that parr, freshwater, and seawater smolts have an alpha(3)-like isoform. This study shows that the increase in Na+-K+-ATP ase activity in smolt gills depends first on an increase in the alpha-mRNA expression and is followed by a slower rise in alpha-protein abundance that eventually leads to st higher synthesis of Na+-K+ pumps.