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

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 bafflomycin-sensitive Na+ uptake. Analysis of the mechanism(s) of Na-22(+) influx was performed in control and acid-activated (addition of 10 mmol 1(-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 nmol 1(-1)) treatment or internal acidification with addition of proprionic acid (10mmol1(-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 (alpha-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 alpha-MR cells function in branchial Na+ uptake in freshwater fish.