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). 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 (alpha-MR cells) demonstrated phenamil-and bafilomycin-sensitive acid-activated (22)Na(+) uptake. We confirm the coupling of a V-type H(+)-ATPase with phenamil-sensitive Na(+) uptake activity and conclude that high-density alpha-MR cells function in branchial Na(+) uptake in freshwater fish.
Reid
Scott D
SD
Dept of Biology, Okanagan University College, Kelowna, British Columbia, VIV 1V7, Canada. sdreid@ouc.bc.ca
Hawkings
G S
GS
Galvez
F
F
Goss
G G
GG
D000584:Amiloride / Q000031:analogs & derivatives* / Q000494:pharmacology*; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects; D002034:Bumetanide / Q000494:pharmacology; D004847:Epithelial Cells / Q000187:drug effects / Q000502:physiology*; D005260:Female; D005880:Gills / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D006146:Guanidines / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D018942:Macrolides; D008297:Male; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D008954:Models, Biological; D017686:Oncorhynchus mykiss / Q000502:physiology*; D010042:Ouabain / Q000494:pharmacology; D019887:Peanut Agglutinin / Q000494:pharmacology; D012964:Sodium / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012979:Sodium Radioisotopes; D013450:Sulfones / Q000494:pharmacology
D000900:Anti-Bacterial Agents; D006146:Guanidines; D018942:Macrolides; D019887:Peanut Agglutinin; D012979:Sodium Radioisotopes; D013450:Sulfones; D002034:Bumetanide; C074107:3-methylsulfonyl-4-piperidinobenzoyl guanidine; C047088:phenylamil; D010042:Ouabain; D000584:Amiloride; C040929:bafilomycin A1; D012964:Sodium