Interactions between [Na.sup.+] channels and [Na.sup.+]-HC[O.sup.-.sub.3] cotransporters in the freshwater fish gill MR cell: a model for transepithelial [Na.sup.+] uptake

Isolated mitochondria-rich (MR) cells from the rainbow trout gill epithelium were subjected to intracellular pH ([pH.sub.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.sub.i] from an N[H.sup.4]Cl-induced acidification in the absence of [Na.sup.+]. An apparent link between resting [pH.sub.i] and [Na.sup.+]-independent [pH.sub.i] recovery was made. We observed a unique [pH.sub.i] acidification event that was induced by extracellular [Na.sup.+] addition. This further classified the mixed MR cell population into two functional subtypes: the majority of cells (77%) demonstrated the [Na.sup.+]-induced [pH.sub.i] acidification, whereas the minority (23%) demonstrated an alkalinization of [pH.sub.i] under the same circumstances. The focus of this study was placed on the [Na.sup.+]-induced acidification and pharmacological analysis via the use of amiloride and phenamil, which revealed that [Na.sup.+] uptake was responsible for the intracellular acidification. Further experiments revealed that [pH.sub.i] acidification could be abolished when [Na.sup.+] was allowed entry into the cell, but the activity of an electrogenic [Na.sup.+]-HC[O.sup.-.sub.3] cotransporter (NBC) was inhibited by DIDS. The electrogenic NBC activity was supported by a DIDS-sensitive, [Na.sup.+]-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.sup.+] uptake occurring via an apical [Na.sup.+] channel linked to a basolateral, electrogenic NBC in one subpopulation of MR cells. epithelial sodium channels; sodium-induced acidification; amiloride; phenamil; DIDS; acid-base; peanut lectin agglutinin binding; membrane potential; BCECF-AM; bis-oxonol; ion uptake