Osmoregulation by gills of euryhaline crabs: Molecular analysis of transporters

American Zoologist, Sep 2001 by Towle, David W, Weihrauch, Dirk

RELATED ENZYMES

Na^sup ^ K^sup ^-ATPase

Originally described enzymatically in homogenates of nerves from the shore crab Carcinus mamas (Skou, 1957), the Na^sup ^ K^sup ^-ATPase is known to increase in activity in gills of aquatic animals when they are osmotically stressed. For example, transferring blue crabs (Callinectes sapidus) from 35% salinity to 5% leads to a doubling of Na^sup ^ K^sup ^-ATPase activity in the ion-transporting posterior gills (Towle et al., 1976). Similar results are seen in the shore crab Carcinus mamas (Siebers et al., 1982) and many other species. These results imply that the energetic costs of osmoregulation in stressful conditions are met, at least in part, by enhanced activity of the Na^sup ^ K^sup -^ATPase.

It has not been clear whether the salinityrelated changes in Na^sup ^ K^sup ^-ATPase activity result from enzyme activation or from enzyme (and perhaps mRNA) synthesis. Western blot analysis of Na^sup ^ K^sup ^-ATPase alpha-subunit protein in plasma membranes prepared from Carcinus mamas gills indicated that crabs acclimated for 3 wk to low salinity showed twice the amount of a-subunit protein compared with crabs acclimated to high salinity or to low salinity for just 4 hr (Lucu and Flik, 1999).

Recent molecular data from our laboratory have revealed a higher abundance of Na KI-ATPase a-subunit mRNA as well as protein in posterior gills of Callinectes sapidus compared to anterior gills. However, we observed little effect of salinity change on either a-subunit mRNA or asubunit protein (Paulsen et al., 2000; Towle et al., 2001). Thus our evidence suggests that tissue differences in Na^sup ^ K^sup ^-ATPase activity in gills of the blue crab are the result of gene regulation, leading to enhanced mRNA and protein levels. Salinity-related changes in Na^sup ^ K^sup ^-ATPase activity, perhaps exerted by hormone-sensitive translational or cell signaling processes, may be superimposed upon the likely differences in gene transcription. In conclusion, molecular properties of the Na^sup ^ K^sup ^-ATPase fulfill two of our criteria for inclusion in a transport model, suggesting a central role of the Na^sup ^ K^sup ^-ATPase in osmoregulatory ion transport.

Vacuolar-type He-ATPase

cDNAs coding for the B subunit of the V-type ATPase have been identified and at least partially sequenced in gills of five brachyuran crab species (Weihrauch et al., 2000). A detailed study of the expression of B subunit mRNA in gills of Carcinus maenas suggests that the V-type H^sup ^ATPase is not expressed preferentially in posterior ion-transporting gills, nor is its expression enhanced by osmoregulatory challenge (Fig. 5) (Weihrauch et al., 2000). The V-type H^sup ^-ATPase thus satisfies the first criterion for inclusion as a candidate transporter in C maenas, but not the second and third. On the other hand, in the freshwatertolerant Chinese crab E. sinensis, V-type H^sup ^-ATPase B subunit mRNA is preferentially expressed in ion-transporting posterior gills of osmotically challenged animals (Weihrauch et al., 2000), thus meeting all three of the proposed criteria for inclusion in transport models. The shore crab C. mamas may not survive in freshwater because it is incapable of activating membrane-specific V-type ATPase gene transcription.