Carbonic anhydrase 2-like a and 15a are involved in acid-base regulation and Na⁺ uptake in zebrafish H⁺-ATPase-rich cells

Abstract

H(+)-ATPase-rich (HR) cells in zebrafish gills/skin were found to carry out Na+ uptake and acid-base regulation through a mechanism similar to that which occurs in mammalian proximal tubular cells. However, the roles of carbonic anhydrases (CAs) in this mechanism in zebrafish HR cells are still unclear. The present study used a functional genomic approach to identify 20 CA isoforms in zebrafish. By screening with whole mount in situ hybridization, only zca2-like a and zca15a were found to be expressed in specific groups of cells in zebrafish gills/skin, and further analyses by triple in situ hybridization and immunocytochemistry demonstrated specific colocalizations of the two zca isoforms in HR cells. Knockdown of zca2-like a caused no change in and knockdown of zca15a caused an increase in H+ activity at the apical surface of HR cells at 24 h postfertilization (hpf). Later, at 96 hpf, both the zca2-like a and zca15a morphants showed decreased H+ activity and increased Na+ uptake, with concomitant upregulation of znhe3b and downregulation of zatp6v1a (H+-ATPase A-subunit) expressions. Acclimation to both acidic and low-Na+ fresh water caused upregulation of zca15a expression but did not change the zca2-like a mRNA level in zebrafish gills. These results provide molecular physiological evidence to support the roles of these two zCA isoforms in Na+ uptake and acid-base regulation mechanisms in zebrafish HR cells.

Ion uptake and acid secretion in zebrafish (Danio rerio)

“…A study by Hirose's group (Esaki et al, 2007) also supported this notion; zCA2-like mRNA was colocalized in Sodium Green-accumulating HR cells, and 10 μmol l -1 ethoxzolamide (a CA inhibitor) was found to abolish 65% of the Sodium Green accumulation in zebrafish HR cells. On the other hand, acclimation to both acidic and low-Na + FW caused upregulation of zCA15a expression but did not change the zCA2-like a mRNA level in zebrafish gills (Lin et al, 2008). Interestingly, knockdown of zCA2-like a caused upregulation of zCA15a expression, while knockdown of zCA15a did not affect the expression of zCA2-like a in zebrafish morphants.…”

“…Morphants of zCA15a had increased H + activity at the apical surface of HR cells at 24 h post-fertilization, while those of zCA2-like a showed no change. Later, at 96 h postfertilization, both zCA15a and zCA2-like a showed decreased H + activity and increased Na + uptake, with concomitant upregulation of zNHE3b and downregulation of zATP6V1a (H + -ATPase Asubunit) expression (Lin et al, 2008), demonstrating the roles of the 2 CA isoforms in the functions of HR cells. A study by Hirose's group (Esaki et al, 2007) also supported this notion; zCA2-like mRNA was colocalized in Sodium Green-accumulating HR cells, and 10 μmol l -1 ethoxzolamide (a CA inhibitor) was found to abolish 65% of the Sodium Green accumulation in zebrafish HR cells.…”

“…Acclimation to low-Na + FW caused upregulation of zNHE3b and downregulation of H + -ATPase, while an acidic environment induced reverse responses, downregulation of zNHE3b and upregulation of H + -ATPase, in zebrafish HR cells (Yan et al, 2007). Knockdown of zCA2-like a or zCA14b also caused stimulation of zNHE3b and suppression of H + -ATPase (Lin et al, 2008). Taking all these findings together, a model for Na + -uptake and acid-base regulation in zebrafish HR cells was proposed (Yan et al, 2007): in low-Na + environments, apical H + -ATPase is downregulated to maintain an intracellular H + gradient to facilitate Na + uptake via apical zNHE3b, which is the dominant player in regulating the internal Na + balance.…”

“…As HR cells were demonstrated to have similar functions of acid-base regulation and Na + uptake, they should also express carbonic anhydrases (CAs) and the basolateral H + -HCO 3 + cotransporter (NBC) to fulfill the transepithelial transport mechanisms, as do proximal tubular cells. Recently, Lin and colleagues (Lin et al, 2008) identified 20 CA isoforms and cloned 10 of them. Triple ISH and ICC results showed that only two isoforms, zCA2-like a and zCA15a, were specifically expressed in HR cells but not in other cells in zebrafish skin/gills.…”

“…However, it was not until the study by Pan and colleagues (Pan et al, 2005) that molecular evidence for the existence of an orthologue of mammalian ECaC in fish skin/gill ionocytes was provided. Using similar approaches to those used in the studies on zebrafish NHE and CA (Yan et al, 2007;Lin et al, 2008), one isoform of ECaC, six isoforms of PMCAs (zPMCA1a, zPMCA1b, zPMCA2, zPMCA3a, zPMCA3b, and zPMCA4) and seven isoforms of NCX (zNCX1a, zNCX1b, zNCX2a, zNCX2b, zNCX3, zNCX4a and zNCX4b) were identified in zebrafish, and triple fluorescence ISH and ICC were conducted to demonstrate the colocalization of zECaC, zPMCA2 and zNCX1b mRNAs in a portion of skin/gill NaR cells (Pan et al, 2005;Liao et al, 2007). Acclimation to a low-Ca 2+ environment caused stimulation of zebrafish Ca 2+ -uptake function and a concomitant upregulation in skin/gill zECaC expression, but no effect on the expression of either zPMCAs or zNCXs (Pan et al, 2005;Liao et al, 2007), supporting the current Ca 2+ -uptake model, in which ECaC may act as a major regulatory target for this mechanism during environmental challenge.…”