Osmoregulation by gills of euryhaline crabs: Molecular analysis of transporters

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

Na^sup ^/H^sup ^ Exchanger

A Na^sup ^/H^sup ^ exchanger cDNA has been amplified and sequenced from gills of two crab species, Carcinus maenas and Callinectes sapidus (Newton et al., 1996; Towle et al., 1997). Closely related to the vertebrate isoforms previously sequenced, it is not known whether the cloned exchanger is identical with the electrogenic exchanger described in membrane vesicles from crustacean epithelia (Shetlar and Towle, 1989). Nevertheless, the cloned exchanger is expressed very strongly at the mRNA level in gills, showing much lower abundance in all other tissues examined (Fig. 6). Thus the Na^sup ^/H^sup ^ exchanger meets the first two criteria for retention as a candidate transporter involved in osmoregulation. Whether exchanger mRNA levels respond to osmoregulatory challenge is the subject of current work.

Na^sup ^/K^sup ^/2Cl^sup -^ cotransporter

A putative Na^sup ^/K^sup ^/2Cl^sup -^cotransporter has been identified and sequenced in gills of blue crab Callinectes sapidus (GenBank Accession Number AF190129) (Towle, 1998). Gills of the shore crab C. maenas also express the Na^sup ^/K^sup ^/2Cl^sup -^ cotransporter (Weihrauch and Towle, unpublished), contrary to earlier reports from our lab. Preliminary evidence suggests that the cotransporter is expressed preferentially in posterior gills of C. sapidus (Towle, 1998) (Fig. 7). Current work is examining whether gills of C. maenas express the cotransporter strongly and also whether osmoregulatory challenge enhances mRNA and protein expression.

Carbonic anhydrase

We have successfully amplified two different isoforms of carbonic anhydrase cDNA from gills of Callinectes sapidus and Carcinus maenas (Gehnrich et al., 2000a, b). Messenger RNA coding for one of the isoforms is much more abundant than the mRNA coding for the second isoform. Both are more abundant in posterior gills than in anterior gills. In addition, the levels of carbonic anhydrase mRNA increase dramatically when crabs are transferred from high to low salinity (Fig. 8). Our evidence suggests therefore that carbonic anhydrase satisfies all three criteria for inclusion as a transport-related enzyme critical for osmoregulation by crab gill, likely enhancing the availability of counterions for transporter proteins.

Arginine kinase

In the course of molecular work on transporters, we serendipitously amplified a cDNA coding for arginine kinase, producing a complete cDNA sequence for Carcinus mamas and a complete open reading frame for Callinectes sapidus (Kotlyar et al., 2000). Although the enzymatic activity of arginine kinase is enhanced following transfer of C. sapidus from high to low salinity, mRNA abundance is not affected by this osmoregulatory challenge. In the weaker osmoregulator C. maenas, both enzyme activity and mRNA abundance were not responsive to salinity reduction (Fig. 5). We can conclude that arginine kinase expression is not tightly coupled to osmoregulatory processes in these two species. The enzyme may, however, mediate buffering of ATP levels that are generally required for active ion transport and other processes.