Isolation and characterization of mitochondria-rich cell types from the gill of freshwater rainbow trout.
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.
Galvez
Fernando
F
Department of Biological Sciences, University of Alberta, Edmonton, Alberta, T6G 2E9, Canada.
Reid
Scott D
SD
Hawkings
Guy
G
Goss
Greg G
GG
D000818:Animals; D002469:Cell Separation / Q000379:methods; D002712:Chlorides / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism / Q000648:ultrastructure; D005880:Gills / Q000166:cytology* / Q000378:metabolism / Q000502:physiology / Q000648:ultrastructure; D008280:Magnetics; D008928:Mitochondria / Q000648:ultrastructure*; D017686:Oncorhynchus mykiss / Q000033:anatomy & histology* / Q000378:metabolism; D019887:Peanut Agglutinin / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000378:metabolism
D002712:Chlorides; D019887:Peanut Agglutinin; D006180:Proton-Translocating ATPases