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

Abstract

Several parameters were analyzed to determine the mechanisms responsible for the enhancement of the gill Na(+)-K(+)-ATPase activity of Atlantic salmon smolts. A major alpha-subunit transcript of 3.7 kb was revealed by Northern blot in both parr and smolt gills when hybridized with two distinct cDNA probes. The alpha-mRNA abundance demonstrated an increase to maximal levels in smolts at an early stage of the parr-smolt transformation. This was followed 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 activity, both only reaching maximum levels a month later, at the peak of the transformation process. Parr fish experienced a decrease in alpha-mRNA abundance and had basal levels of alpha-protein and enzyme activity. Measurement of the binding of [(3)H]ouabain to Na(+)-K(+)-ATPase was characterized in smolts and parr gill membranes showing more than a twofold elevation in smolts 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 levels 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-protein was performed with polyclonal antibodies directed against the rat alpha-specific isoforms, revealing that parr, freshwater, and seawater smolts have an alpha(3)-like isoform. This study shows that the increase in Na(+)-K(+)-ATPase 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 a higher synthesis of Na(+)-K(+) pumps.