Interactions between Na+ channels and Na+-HCO3- cotransporters in the freshwater fish gill MR cell: a model for transepithelial Na+ uptake.
Isolated mitochondria-rich (MR) cells from the rainbow trout gill epithelium were subjected to intracellular pH (pH(i)) imaging with the pH-sensitive dye BCECF-AM. MR cells were categorized into two distinct functional subtypes based on their ability to recover pH(i) from an NH(4)Cl-induced acidification in the absence of Na(+). An apparent link between resting pH(i) and Na(+)-independent pH(i) recovery was made. We observed a unique pH(i) acidification event that was induced by extracellular Na(+) addition. This further classified the mixed MR cell population into two functional subtypes: the majority of cells (77%) demonstrated the Na(+)-induced pH(i) acidification, whereas the minority (23%) demonstrated an alkalinization of pH(i) under the same circumstances. The focus of this study was placed on the Na(+)-induced acidification and pharmacological analysis via the use of amiloride and phenamil, which revealed that Na(+) uptake was responsible for the intracellular acidification. Further experiments revealed that pH(i) acidification could be abolished when Na(+) was allowed entry into the cell, but the activity of an electrogenic Na(+)-HCO(3)(-) cotransporter (NBC) was inhibited by DIDS. The electrogenic NBC activity was supported by a DIDS-sensitive, Na(+)-induced membrane potential depolarization as observed via imaging of the voltage-sensitive dye bis-oxonol. We also demonstrated NBC immunoreactivity via Western blotting and immunohistochemistry in gill tissue. We propose a model for transepithelial Na(+) uptake occurring via an apical Na(+) channel linked to a basolateral, electrogenic NBC in one subpopulation of MR cells.
Parks
Scott K
SK
Dept. of Biological Sciences, Univ. of Alberta, Edmonton, Alberta, T5G 2E9, Canada. skparks@ualberta.ca
Tresguerres
Martin
M
Goss
Greg G
GG
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001639:Bicarbonates / Q000378:metabolism; D002470:Cell Survival; D004847:Epithelial Cells / Q000378:metabolism*; D005452:Fluoresceins; D005456:Fluorescent Dyes; D005880:Gills / Q000166:cytology / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D015640:Ion Channel Gating; D017136:Ion Transport; D008564:Membrane Potentials; D008928:Mitochondria / Q000378:metabolism; D023421:Models, Animal; D017686:Oncorhynchus mykiss; D012964:Sodium / Q000378:metabolism*; D015222:Sodium Channels / Q000502:physiology*; D027982:Sodium-Bicarbonate Symporters / Q000502:physiology*
D001639:Bicarbonates; D005452:Fluoresceins; D005456:Fluorescent Dyes; D015222:Sodium Channels; D027982:Sodium-Bicarbonate Symporters; C057433:2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester; C047088:phenylamil; D000584:Amiloride; D012964:Sodium; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid