A magnetic cell separation technique (MACS) was developed for isolating and characterizing peanut lectin agglutinin positive (PNA(+)) cells from rainbow trout gills. Percoll density separated mitochondria-rich (MR) cells were serially labeled with PNA-FITC and an anti-FITC antibody covalently coupled to a 50-nm iron particle and then applied to a magnetic column. PNA(+) MR cells were enriched to >95% purity. Transmission electron microscopy analysis of both the PNA(+) and PNA negative (PNA(-)) fraction showed that PNA binds to MR chloride cells while the PNA(-) cell fraction is comprised of MR cells with features characteristic of pavement cells. Western blotting demonstrated that both PNA(+) and PNA(-) fractions had high levels of Na(+)-K(+)-ATPase and Sco1 expression; however, relative expression of H(+)-ATPase in PNA(+) and PNA(-) cells demonstrated that untreated fish had twofold higher H(+)-ATPase levels in PNA(-) cells relative to the PNA(+) cells. Furthermore, hypercapnic acidosis significantly increased the relative H(+)-ATPase expression on PNA(-) cells only, whereas metabolic alkalosis had no significant effect.

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.

SUMMARYMitochondria-rich cells (MR cells) of the gills of rainbow trout undergo changes in relative distribution and biochemical function during acclimation to partial-strength (10‰) and full-strength (30‰) seawater. In isolated total gill cells, Na+/K+-ATPase activity increased fivefold and H+-ATPase activity decreased fourfold when trout were acclimated to either 10‰ or 30‰ seawater. When total MR gill cells were separated based on differential binding to peanut lectin agglutinin (PNA), the PNA subtypes underwent a change in relative distribution in seawater-acclimated fish. In freshwater, the ratio of PNA–:PNA+ was 65:35 while in seawater the distribution changed to 20:80 PNA–:PNA+. Additionally, differential changes in Na+/K+-ATPase and H+-ATPase activity in each of the independent cell types occurred during seawater acclimation; Na+/K+-ATPase activity in the PNA– cells increased by 197% while in PNA+cells Na+/K+-ATPase decreased by 57%. However,H+-ATPase activity was decreased in both PNA–(84%) and PNA+ (72%) subtypes during acclimation to seawater.

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