Carbonic Anhydrase in the Gills of Seawater-and Freshwater-acclimated Flounders Platichthys flesus: Purification, Characterization, and Immunohistochemical Localization

Abstract

SUMMARYFlounders Platichthys flesus were investigated with respect to isolation, purification, and cellular localization of carbonic anhydrase (CA) in the respiratory system. CA was purified from gills and erythrocytes and was shown to exclusively represent a soluble enzyme with an apparent molecular weight of 30 kD. Inhibition constants (K I ) towards acetazolamide (ACTZ) were 8.4·10 Ϫ 9 M for erythrocyte CA and 7.6·10 Ϫ 9 M for gill CA, indicating a high sensitivity to sulfonamides, as exhibited by human CA II. Specific CA activity did not differ significantly in seawater-and freshwater-acclimated fish. Antibodies were raised against purified gill and erythrocyte CA. Both antisera crossreacted and were used to localize CA in the gills of seawater and freshwater flounders at the light microscopic level. Independent of the salinity, a positive reaction of variable intensity was found in the following cell types: pavement cells (PVCs), forming the gill epithelial surface layer; mucous cells (MCs); pillar cells (PCs), bordering the vascular channels of the secondary lamellae; and chloride cells (CCs), mitochondria-rich cells located in the primary epithelium, the interlamellar regions, and at the bases of the secondary lamellae.

The role of branchial carbonic anhydrase in acid-base regulation in rainbow trout (Oncorhynchus mykiss)

“…Immunocytochemical localization of branchial CA using an antibody raised against purified trout gill CA confirmed the broad distribution reported by Conley and Mallatt (1988) and additionally indicated that CA was concentrated in the apical region of both pavement cells and MR cells (Rahim et al, 1988). However, these patterns are not true of all species examined to date; in some species CA appears to be confined to either pavement cells or MR cells, while in others it is found in both (Dimberg et al, 1981;Conley and Mallatt, 1988;Flügel et al, 1991;Sender et al, 1999;Wilson et al, 2000b,c;Hirata et al, 2003;Choe et al, 2004).…”

“…evidence indicates that branchial cytoplasmic CA is present in both pavement and MR cells, with a generally apical location (Rahim et al, 1988;Sender et al, 1999;Wilson et al, 2000b). Rahim et al (1988) also provided immunological evidence for the presence of distinct branchial and blood CA isoforms in rainbow trout and carp, a finding that was recently confirmed by the cloning of rainbow trout blood (tCAb) and cytosolic (tCAc) CA isoforms (Esbaugh et al, 2004(Esbaugh et al, , 2005.…”

Cytoplasmic carbonic anhydrase isozymes in rainbow troutOncorhynchus mykiss: comparative physiology and molecular evolution

“…Branchial CA activity is thought to be largely cytoplasmic, occurring primarily in pavement cells and chloride cells (Lacy, 1983;Conley and Malalatt, 1988;Rahim et al, 1988;Flügel et al, 1991;Sender et al, 1999;Wilson et al, 2000), with the exception of membrane bound isozymes found in the gills of elasmobranchs (Swenson and Maren, 1987;Gilmour et al, 1997Gilmour et al, , 2001Gilmour et al, , 2002Henry et al, 1997;Wilson et al, 2000), and Antarctic fishes, Chaenocephalus aceratus and Notothenia coriiceps . Evidence is mixed on whether the same CA isozyme ·The Journal of Experimental Biology 208, 1951Biology 208, -1961 Published by The Company of Biologists 2005Biologists doi:10.1242 It is well established that the gills of teleost fish contain substantial levels of cytoplasmic carbonic anhydrase (CA), but it is unclear which CA isozyme(s) might be responsible for this activity.…”

Characterization of the Column and Autocellular Junctions That Define the Vasculature of Gill Lamellae

“…Recently, the presence of carbonic anhydrase (Sender et al 1999;Gilmour et al 2007), ammonia transporter (Nakada et al 2007), and a member of the winged helix/forkhead transcription factor FoxF1 (Hidaka et al 2004), which is required for normal development of lung and foregut in mammals (Mahlapuu et al 2001), have been demonstrated in pillar cells. These observations, as well as the results of this study, have highlighted the importance of pillar cells in respiration and nitrogen metabolism.…”