Journal
AMERICAN JOURNAL OF PHYSIOLOGY-REGULATORY INTEGRATIVE AND COMPARATIVE PHYSIOLOGY
Volume 282, Issue 3, Pages R658-R668Publisher
AMER PHYSIOLOGICAL SOC
DOI: 10.1152/ajpregu.00342.2001
Keywords
peanut lectin; pavement cell; fish; hypercapnia; hydrogen-adenosine-triphosphatase
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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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