Isolation and characterization of mitochondria-rich cell types from the gill of freshwater rainbow trout

Abstract

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.