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

Scott D. Reid^1,2,*, G. S. Hawkings², F. Galvez² and G. G. Goss²

¹ Dept of Biology, Okanagan University College, Kelowna, British Columbia, VIV 1V7, Canada
² Dept of Biological Sciences, University of Alberta, Edmonton, Alberta, T5G 2E9, Canada

View larger version (18K): [in a new window]   Fig. 1. Relative distribution of isolated gill epithelial cells. Cells were separated into different fractions using a discontinuous Percoll density gradient, peanut lectin agglutinin (PNA) binding and magnetic separation (see Materials and methods). Insert shows the numbers of the PNA- and PNA+ cells found within the 1.05-1.09 g ml-1 density interface layers expressed as a percentage of the total epithelial cells harvested. Data represent means + 1 S.E.M. (N=4). Means with different letters indicate that they are significantly different (P0.05).

View larger version (19K): [in a new window]   Fig. 2. The effect of the Na+/H+-exchange inhibitor HOE-694 alone or in combination with propionate and phenamil on Na+ influx rates of trout gill cells. Gill epithelial cells were collected at a 1.03-1.09 g ml-1 Percoll density interface layer, as described in the Materials and methods, and Na+ influx rates were conducted in low (15 mmol l-1) Na+ saline to which 50 µmol l-1 ouabain and 20 µmol l-1 bumetanide were added (see Materials and methods). The open histogram represents the Na+ influx rate from control (pH 7.8) gill cells in the absence of HOE-694. The shaded (gray) histogram represents the Na+ influx rate from cells incubated in the presence of 50 µmol l-1 HOE-694. The hatched histogram represents the Na+ influx rate for control gill cells in the presence of HOE-694 and 10 mmol l-1 Na+ proprionate. The filled (black) histogram represents the Na+ influx rate for cells in the presence of HOE-694, proprionate and 10 µmol l-1 phenamil. Data represent means + 1 S.E.M. (N=4). Means with different letters indicate that they are significantly different (P0.05).

View larger version (15K): [in a new window]   Fig. 3. Na+ uptake in cells isolated from the 1.03-1.05 g ml-1 interface of the Percoll density gradient. Na+ influx rates were conducted as described in Materials and methods. Na+ influx rates were determined in the absence of any additional inhibitors (control; open histograms), 10 µmol l-1 phenamil (Phen; shaded histograms), 10 nmol l-1 bafilomycin A1 (Baf; hatched histograms) or a combination of phenamil and bafilomycin (Phen + Baf; filled histograms) and in either the absence (unstimulated) or presence (stimulated) of 10 mmol l-1 Na+ proprionate. Data represent means + 1 S.E.M. (N=3). Means with different letters indicate that they are significantly different (P0.05).

View larger version (20K): [in a new window]   Fig. 4. Na+ uptake in cells isolated from the 1.05-1.09 g ml-1 interface of the Percoll density gradient. Na+ influx rates were conducted as described in Materials and methods. Na+ flux rates were determined in the absence of any additional inhibitors (control; open histograms), 10 µmol l-1 phenamil (Phen; shaded histograms), 10 nmol l-1 bafilomycin A1 (Baf; hatched histograms) or a combination of phenamil and bafilomycin (Phen + Baf; filled histograms) and in either the absence (unstimulated) or presence (stimulated) of 10 mmol l-1 Na+ proprionate. Data represent means + 1 S.E.M. (N=3). Means with different letters indicate that they are significantly different (P0.05).

View larger version (19K): [in a new window]   Fig. 5. Na+ uptake in PNA+ mitochondria-rich gill cells of rainbow trout. Gill epithelial cells were collected from the 1.05-1.09 g ml-1 Percoll density interface and further isolated using lectin binding and magnetic cell separation. Na+ influx rates were conducted as described in Materials and methods. Na+ influx rates were determined in the absence of any additional inhibitors (control; open histograms), 10 µmol l-1 phenamil (Phen; shaded histograms), 10 nmol l-1 bafilomycin A1 (Baf; hatched histograms) or a combination of phenamil and bafilomycin (Phen + Baf; filled histograms) and in either the absence (unstimulated) or presence (stimulated) of 10 mmol l-1 Na+ proprionate. Data represent means + 1 S.E.M. (N=3). Means with different letters indicate that they are significantly different (P0.05).

View larger version (18K): [in a new window]   Fig. 6. Na+ uptake in PNA- mitochondria-rich gill cells of rainbow trout. Gill epithelial cells were collected from the 1.05-1.09 g ml-1 Percoll density interface and further isolated using lectin binding and magnetic cell separation. Na+ influx rates were conducted as described in Materials and methods. Na+ influx rates were determined in the absence of any additional inhibitors (control; open histograms), 10 µmol l-1 phenamil (Phen; shaded histograms), 10 nmol l-1 bafilomycin A1 (Baf; hatched histograms) or a combination of phenamil and bafilomycin (Phen + Baf; filled histograms) and in either the absence (unstimulated) or presence (stimulated) of 10 mmol l-1 Na+ proprionate. Data represent means + 1 S.E.M. (N=3). Means with different letters indicate that they are significantly different (P0.05).