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

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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.

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American Journal of Physiology - Regulatory Integrative and Comparative Physiology

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