Localization and characterization of phenamil-sensitive Na+influx in isolated rainbow trout gill epithelial cells

Abstract

SUMMARYPercoll density-gradient separation, combined with peanut lectin agglutinin(PNA) binding and magnetic bead separation, was used to separate dispersed fish gill cells into sub-populations. Functional characterization of each of the sub-populations was performed to determine which displayed acid-activated phenamil- and bafilomycin-sensitive Na+ uptake. Analysis of the mechanism(s) of 22Na+ influx was performed in control and acid-activated (addition of 10 mmoll-1 proprionic acid) cells using a variety of Na+ transport inhibitors (ouabain, phenamil,HOE-694 and bumetanide) and a V-type ATPase inhibitor (bafilomycin). We found that cells migrating to a 1.03-1.05 g ml-1 Percoll interface[pavement cells (PVCs)] possessed the lowest rates of Na+ uptake and that influx was unchanged during either bafilomycin (10 nmoll-1) treatment or internal acidification with addition of proprionic acid (10 mmoll-1). Mitochondria-rich (MR) cells that migrated to the 1.05-1.09 g ml-1 interface of the Percoll gradient demonstrated acidification-activated bafilomycin and phenamil-sensitive Na+ influx. Further separation of the MR fraction into PNA+ and PNA- fractions using magnetic separation demonstrated that only the PNA- cells (α-MR cells)demonstrated phenamil-and bafilomycin-sensitive acid-activated 22Na+ uptake. We confirm the coupling of a V-type H+-ATPase with phenamil-sensitive Na+ uptake activity and conclude that high-density α-MR cells function in branchial Na+ uptake in freshwater fish.

Ion uptake and acid secretion in zebrafish (Danio rerio)

“…However, several studies on other species including rainbow trout favoured the operation of ENaC-like channels in the apical Na + -uptake pathway. Goss and colleagues (Reid et al, 2003;Parks et al, 2007) Fig. 1.…”

“…PNA -and PNA + MR cells, which were isolated with PNA, express higher H + -ATPase and NKA, respectively (Galvez et al, 2002), and show different ion-transport functions: PNA -exhibited a bafilomycin- sensitive acid-activated 22 Na + -uptake/acid-stimulated response of H + -ATPase, while PNA + showed Ca 2+ uptake (Reid et al, 2003;Galvez et al, 2006;Parks et al, 2007). Based on many previous immunocytochemical, physiological and pharmacological data, the current model for trout gill cells proposes that PNA -and PNA + MR cells are responsible for Na + uptake/acid secretion and Ca 2+ uptake/base secretion/Cl -uptake, respectively (Tresguerres et al, 2006;Perry and Gilmour, 2006;Evans, 2008;Parks et al, 2008).…”

Pharmacological characterisation of apical Na+ and Cl– transport mechanisms of the anal papillae in the larval mosquitoAedes aegypti

“…A0554 and D3514). The doses of the inhibitors were selected based on previous invertebrate or vertebrate tissue studies such that they were selective for their intended targets [phenamil (Reid et al, 2003;Kleyman and Cragoe, 1988); ouabain (MacVicker et al, 1993); bafilomycin (Beyenbach et al, 2000); methazolamide, DIDS and SITS (Smith et al, 2010;Nguyen and Donini, 2010); HMA (Kleyman and Cragoe, 1988;Tsui et al, 2009)]. All inhibitors were dissolved in DMSO to yield stock solutions that were subsequently diluted in bathing solution as described above.…”

Isolation and fractionation of gill cells from freshwater (Lasmigona costata) and seawater (Mesodesma mactroides) bivalves for use in toxicological studies with copper

“…-ATPase activity. The two cell fractions were more equal in M. mactroides (Fraction I: 51.7 ± 3.2 %; Fraction II: 48.3 ± 3.2 %) but still not comparable with fish results (Reid et al, 2003;Galvez et al 2008). Probably, this is a consequence of the less aggressive mechanical method applied for cell dissociation.…”

“…functional characterization of cell membrane transporters related to Na ? uptake (Goss et al 2001;Galvez et al 2002;Reid et al 2003), and particularly in analyzing the ionoregulatory disrupting effects of metals such as Cu (Goss et al 2011). Cu is now generally recognized to be an ionoregulatory toxicant in aquatic invertebrates (Pinho et al 2007;Lopes et al 2011a, b) as well as in fish.…”