Salinity-stimulated changes in expression and activity of two carbonic anhydrase isoforms in the blue crabCallinectes sapidus

Abstract

Two isoforms of the enzyme carbonic anhydrase (CA) in the blue crab gill, CasCAg and CasCAc, were identified, sequenced, and found to match the membrane-associated and cytoplasmic isoforms, respectively. The membrane-associated isoform is present in much higher levels of mRNA expression in both anterior and posterior gills in crabs acclimated to high salinity (35 p.p.t.), but expression of the cytoplasmic isoform in the posterior gill undergoes a significantly greater degree of up-regulation after exposure to low salinity (15 p.p.t.). CasCAc has the largest scope of induction (100-fold) reported for any transport-related protein in the gill, and this may be necessary to overcome diffusion limitations between gill cytoplasm and the apical boundary layer. Furthermore, the timing of the changes in expression of CasCAc corresponds to the timing of the induction of protein-specific CA activity and CA protein concentration. No changes in CA mRNA expression or activity occur in the anterior gills. The pattern of up-regulation of expression of mRNA of the alpha-subunit of the Na+/K+-ATPase is similar to that for CasCAc, and both precede the establishment of the new acclimated physiological state of the crab in low salinity. A putative ;housekeeping' gene, arginine kinase, also showed about a threefold increase in expression in response to low salinity, but only in the posterior gills. These results suggest that for studies of expression in crustacean gill tissue, a control tissue, such as the anterior gill, be used until an adequate control gene is identified.