H+ -ATPase Activity in Crude Homogenates of Fish Gill Tissue: Inhibitor Sensitivity and Environmental and Hormonal Regulation

Abstract

N-ethymaleimide-sensitive ATPase activity was measured in crude homogenates of gill tissue from rainbow trout using a coupled-enzyme ATPase assay in the presence of EGTA, ouabain and azide. This NEM-sensitive ATPase activity, determined to be about 1.5 micromole mg-1 protein h-1 at 15 °C for freshwater trout, is also inhibited by other H+-ATPase blockers such as DCCD, DES, PCMBS and bafilomycin. It is concluded, therefore, that the NEM-sensitive ATPase activity was generated by a proton-translocating ATPase. Since this NEM-sensitive ATPase was also sensitive to the plasma membrane ATPase inhibitor vanadate, we conclude that the H+-ATPase in fish gill is of the plasma membrane type. The major role of the H+-ATPase in the gill epithelium is to facilitate Na+ uptake from fresh water. Sodium concentration in the external medium was the primary regulator of the H+-ATPase in fish gills, with low sodium levels being associated with high H+-ATPase activity. High external calcium concentration had a marked stimulatory effect on H+-ATPase activity in fish gills when the sodium level was low. Environmental hypercapnia induced a 70 % increase in the H+-ATPase activity in fish gills. H+-ATPase activity was also elevated in freshwater fish after chronic cortisol infusion.

Immunochemical analysis of the vacuolar proton-ATPase B-subunit in the gills of a euryhaline stingray (Dasyatis sabina): effects of salinity and relation to Na+/K+-ATPase

“…2). This effect of salinity on branchial V-H + -ATPase expression has also been reported in a teleost (Oncorhynchus mykiss), where gill V-H + -ATPase activity and immunoreactivity decreased when freshwater trout were acclimated to sea water (Lin and Randall, 1993;Lin et al, 1994). A lower branchial V-H + -ATPase expression for the seawater-acclimated stingrays was expected, because active NaCl uptake would not be necessary in a seawater environment and passive Na + /H + and Cl − /HCO3 − exchangers are thought to be responsible for acid-base extrusion in seawater fishes (Claiborne, 1998).…”

“…Since an apical V-H + -ATPase in freshwater teleost gills would directly pump protons into the environment, this transporter has also been hypothesized to play an important role in systemic acid-base balance. Experiments on rainbow trout have corroborated this hypothesis by demonstrating that gill V-H + -ATPase activity, immunoreactivity and mRNA expression all increase after exposure to environmental hypercapnia (Lin and Randall, 1993;Lin et al, 1994;Sullivan et al, 1995;Sullivan et al, 1996;Perry et al, 2000). Studies on branchial V-H + -ATPase in a true marine teleost have yet to be published, but the transporter's role in acid-base regulation of seawater teleosts is assumed to be minimal, given the favorable Na + gradient for Na + /H + exchangers (see Claiborne, 1998;Claiborne et al, 1999).…”

“…In rainbow trout (Oncorhynchus mykiss), V-H + -ATPase and ENaC were expressed in both pavement and chloride (Na + /K + -ATPaserich) cells (Wilson et al, 2000). Other supporting evidence for the proposed role of V-H + -ATPase in freshwater teleost ion uptake includes bafilomycin inhibition of Na + uptake in tilapia (O. mossambicus) and carp (Cyprinus carpio) (Fenwick et al, 1999), and decreased activity and localization of branchial V-H + -ATPase when freshwater rainbow trout are acclimated to sea water (Lin and Randall, 1993;Lin et al, 1994).…”

“…Recently, the V-H + -ATPase has also been considered important for driving Na + uptake across the gill epithelium of freshwater fishes. Similar to processes in frog skin, it was proposed that an apical V-H + -ATPase would generate a favorable electrical gradient to drive Na + uptake through an apical Na + channel (Lin and Randall, 1993;Lin et al, 1994;Sullivan et al, 1995). This model of ion uptake in freshwater teleosts was supported by the results of Wilson et al (Wilson et al, 2000), who found colocalization of the V-H + -ATPase with the epithelial Na + channel (ENaC) on the apical membrane of pavement cells from the leading edge of tilapia gills (Oreochromis mossambicus).…”

Cortisol affects metabolic and ionoregulatory responses to a different extent depending on feeding ration in common carp, Cyprinus carpio

“…Na + /K + ATPase (NKA) activity was measured according to the method described by McCormick (1993) and H + ATPase activity was measured as described by Lin and Randall (1993) modified by Nawata et al (2007) 1) and corticoid receptors (GR1, GR2 and MR) were the same as used by Stolte et al (2008a) (Table 2).…”