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Percoll 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 phenamiland 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.

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Journal of Experimental Biology

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