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The Journal of Experimental Biology 206, 551-559 (2003)
doi: 10.1242/jeb.00109

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

^* Author for correspondence (e-mail: sdreid{at}ouc.bc.ca)

Accepted 28 October 2002

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 ²²Na⁺ 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). We found that cells migrating to a 1.03-1.05 g ml^-1 Percoll interface [pavement cells (PVCs)] possessed the lowest rates of Na⁺ uptake and that influx was unchanged during either bafilomycin (10 nmoll^-1) treatment or internal acidification with addition of proprionic acid (10 mmoll^-1). Mitochondria-rich (MR) cells that migrated to the 1.05-1.09 g ml^-1 interface of the Percoll gradient demonstrated acidification-activated bafilomycin and phenamil-sensitive Na⁺ influx. Further separation of the MR fraction into PNA⁺ and PNA^- fractions using magnetic separation demonstrated that only the PNA^- cells (-MR cells) demonstrated phenamil-and bafilomycin-sensitive acid-activated ²²Na⁺ uptake. We confirm the coupling of a V-type H⁺-ATPase with phenamil-sensitive Na⁺ uptake activity and conclude that high-density -MR cells function in branchial Na⁺ uptake in freshwater fish.

Key words: mitochondria-rich cells, MR, chloride cells, CC, pavement cells, PVC, peanut lectin agglutinin, PNA, transport, fish, Na⁺ influx, density gradient

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