Mechanism of sodium uptake in PNA negative MR cells from rainbow trout, Oncorhynchus mykiss as revealed by silver and copper inhibition.

The rate of acid-stimulated and phenamil-sensitive sodium (Na(+)) uptake was measured in three different cell lineages: pavement cells (PVC), total mitochondrion-rich (MR) cell populations, and peanut lectin agglutinin-negative mitochondrion-rich cells (PNA(-) MR) isolated from the rainbow trout gill epithelium. Despite the presence of basal levels of Na(+) uptake in PVC, this transport was not enhanced by acidification, nor was it inhibited by independent treatment with bafilomycin (i.e.

Seawater acclimation causes independent alterations in Na+/K+- and H+-ATPase activity in isolated mitochondria-rich cell subtypes of the rainbow trout gill.

Mitochondria-rich cells (MR cells) of the gills of rainbow trout undergo changes in relative distribution and biochemical function during acclimation to partial-strength (10 per thousand ) and full-strength (30 per thousand ) seawater. In isolated total gill cells, Na(+)/K(+)-ATPase activity increased fivefold and H(+)-ATPase activity decreased fourfold when trout were acclimated to either 10 per thousand or 30 per thousand seawater. When total MR gill cells were separated based on differential binding to peanut lectin agglutinin (PNA), the PNA subtypes underwent a change in relative distribution in seawater-acclimated fish.

Localization and characterization of phenamil-sensitive Na+ influx in isolated rainbow trout gill epithelial cells.

Percoll density-gradient separation, combined with peanut lectin agglutinin (PNA) binding and magnetic bead separation, was used to separate dispersed fish gill cells into sub-populations. Functional characterization of each of the sub-populations was performed to determine which displayed acid-activated phenamil- and bafilomycin-sensitive Na(+) uptake. Analysis of the mechanism(s) of (22)Na(+) influx was performed in control and acid-activated (addition of 10 mmoll(-1) proprionic acid) cells using a variety of Na(+) transport inhibitors (ouabain, phenamil, HOE-694 and bumetanide) and a V-type ATPase inhibitor (bafilomycin).

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.