Osmoregulation by gills of euryhaline crabs: Molecular analysis of transporters

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

A second candidate for an apical mediator of Na uptake is the Na^sup ^/K^sup ^/2Cl^sup -^ cotransporter, a membrane protein that has been identified in basolateral membranes of salt-excreting epithelia and in apical membranes of salt-absorbing epithelia (Haas, 1994; Russell, 2000). This electroneutral transporter moves Na^sup ^, K^sup ^, and 2Cl^sup -^ ions from extracellular fluid to the cytosol, depending usually on an electrochemical gradient for Na^sup ^. Experiments with gills isolated from the shore crab Carcinus mamas have indicated that a portion of Na^sup ^ uptake from the medium may be coupled to Cl uptake, although little effect of the Na^sup ^/K^sup ^/ 2Cl^sup -^ cotransporter inhibitor furosemide could be demonstrated, possibly due to inhibitor interference by the cuticle (Riestenpatt et al., 1996). Although it is unlikely that the electrochemical Na gradient would be sufficient to drive ion uptake from fresh water via the cotransporter, the cotransporter may play a role in brackish environments inhabited by species like the shore crab.

Two intracellular enzymes that may support active ion transport across crustacean gills are carbonic anhydrase and arginine kinase. Carbonic anhydrase is believed to provide the counterions H^sup ^ and HCO^sub 3^^sup -^ for Na^sup ^ and Cl^sup -^ exchange by catalyzing the hydration of CO2 within gill cells (reviewed by Henry, 1996). The activity of the cytoplasmic form of carbonic anhydrase increases dramatically in gills of euryhaline crabs upon transfer from high to low salinities (Henry, 1988), apparently enhancing the supply of counterions for NaCl uptake.

In the tissues of many invertebrates, arginine kinase catalyzes the hydrolysis of phosphoarginine, phosphorylating adenosine diphosphate to form ATP ATP-utilizing processes such as active ion transport may depend on the ATP buffering function of arginine kinase or on its mitochondrionto-cytosol shuttle function (Ellington and Hines, 1991). Following transfer of crabs from high to low salinities, the enzymatic activity of cytosolic arginine kinase approximately doubles in gills of the strong osmoregulator Callinectes sapidus, but does not change in the more limited osmoregulator Carcinus maenas (Kotlyar et al., 2000).

MOLECULAR ANALYSIS OF TRANSPORTERS

Our approach to characterizing candidate transporters and transport-related enzymes depends on the application of the techniques of molecular biology. To qualify for inclusion in a working model of osmoregulatory ion transport, the following questions should be answered:

(1) Is the candidate cDNA identifiable, in our case by reverse transcription and polymerase chain reaction (RT/PCR)? Although this question may seem simplistic in relation to model building, a clear absence of a particular transporter cDNA would lead to eliminating that transporter from the model.

(2) Is the candidate mRNA expressed preferentially in ion-transporting gills? Comparisons with tissues less specialized for osmoregulatory ion transport may identify transporters that are highly expressed in gills.