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1 Biological Sciences, University of Alberta, Edmonton, Canada
* To whom correspondence should be addressed. E-mail: greg.goss{at}ualberta.ca.
Isolated mitochondria-rich (MR) cells from the rainbow trout gill epithelium were subjected to intracellular pH (pHi) imaging using the pH sensitive dye BCECF-AM. MR cells were categorized into two distinct functional subtypes based on their ability to recover pHi from an NH4Cl induced acidification in the absence of Na+. An apparent link between resting pHi and Na+-independent pHi recovery was made. We observed a unique pHi acidification event that was induced by extracellular Na+ addition. This further classified the mixed MR cell population into two functional subtypes as the majority of cells (77%) demonstrated the Na+-induced pHi acidification while the minority (23%) demonstrated an alkalinization of pHi under the same circumstances. The focus of the study was placed on the Na+ induced acidification and pharmacological analysis via the use of amiloride and phenamil revealed that Na+ uptake was responsible for the intracellular acidification. Further experiments revealed that cellular acidification could be abolished when the activity of an electrogenic Na+/HCO3- cotransporter (NBC) was inhibited by DIDS, even when Na+ was allowed entry into the cell. 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 demonstrate NBC immunoreactivity via western blotting and immunohistochemistry in gill tissue. We proposed a model for transepithelial Na+ uptake occurring via an apical Na+ channel linked to a basolateral outward-directed electrogenic NBC.
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