Mechanisms of Acid Secretion and Sodium Uptake in H+-ATPase-rich (HR) Cell of Larval Zebrafish
Creators: name: Shir-Mohammadi, Khatereh
Publisher: name: Université d'Ottawa / University of Ottawa
Year: 2019
Subjects: Zebrafish
HR cells
Ionocyes

description: Freshwater (FW) fish inhabit hypotonic environments that can vary markedly both spatially and temporally with respect to ambient salt levels and pH. Despite the chemical variability of FW, fish maintain ionic homeostasis (ionic regulation) and pH homeostasis (acid-base regulation) by manipulating ion transport mechanisms within ion-transporting cells (ionocytes) localised to the gills of adults and the skin of larvae. Ionocytes are mitochondrion-rich (MR) cells that, depending on subtype, express specific ion transporters that facilitate the movement of salts and acid-base equivalents across the gills or skin. In zebrafish (Danio rerio), one of the most well-studied ionocytes is termed the H+-ATPase-rich (HR) cell, which is presumed to be a significant site of transepithelial Na+ uptake/acid-secretion. Proteins that have been found in fish zebrafish HR cells include the Na+/H+ exchanger (NHE3), carbonic anhydrases (CA17a and CA15a), proton ATPase (H+-ATPase) and the ammonia channel, ammonia conducting rhesus C glycoprotein b (Rhcgb), which are all thought to function in Na+ uptake acid–base regulation. Ionic and acid-base regulation are achieved both by adjustments to the activity level of these ion transport proteins, but also by regulating the numbers of specific ionocyte subtypes (e.g. HR cells) during acclimation to environments differing in ionic composition or pH. In previous studies, the quantitative assessment of mRNA levels for genes involved in ionic and acid-base regulation relied on measurements using homogenates derived from whole body (larvae) or gill (adult). Such studies cannot distinguish whether any differences in gene expression arise from adjustments of ionocyte subtype numbers, or transcriptional regulation within individual ionocytes. Surprisingly, there are no data on ionocyte-specific gene expression responses in zebrafish exposed to varying environments including acidic or Na+-deficient water. To rectify this gap in the current knowledge, this thesis utilized the florescence activated cell sorting (FACS) approach to separate the HR cells from other cellular sub-populations. The technique was used to measure the gene expression of several HR cell specific transporters and enzymes in isolated HR cells from zebrafish larvae exposed to low pH (pH 4.0) or low Na+ (5 μM) conditions. The data suggest that treatment of larvae at 4 days post fertilization with acidic water caused an increase in h+atpase, ca17a, ca15a, nhe3b and rhcgb mRNA levels in sorted HR cells. These observations suggest the existence of multiple mechanisms of acid secretion in HR cells of larval zebrafish in acidic water; one in which acid secretion via NHE3b is linked to ammonia excretion via Rhcgb, and another facilitated by H+-ATPase. Furthermore, these results provide molecular evidence to support roles for the CA isoforms in acid-base regulation in HR cells. On the other hand, the low Na+ treatment data suggest that nhe3b and rhcgb are the dominant genes maintaining Na+homeostasis. In summary, the results of this thesis demonstrate that acclimation to low pH or low low Na+ environmental conditions is facilitated by HR cell proliferation and HR cell-specific transcriptional control.