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Am J Physiol Regul Integr Comp Physiol 282: R658-R668, 2002; doi:10.1152/ajpregu.00342.2001
0363-6119/02 $5.00
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Vol. 282, Issue 3, R658-R668, March 2002

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

Fernando Galvez1, Scott D. Reid2, Guy Hawkings1, and Greg G. Goss1

1 Department of Biological Sciences, University of Alberta, Edmonton, Alberta, T6G 2E9 Canada; and 2 Department of Biology, Okanagan University College, Kelowna, British Columbia V1V 1V7, Canada

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.

peanut lectin; pavement cell; fish; hypercapnia; hydrogen-adenosine-triphosphatase


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