A Missing Link in the Ionoregulatory Strategy of Larval Sea Lamprey (Petromyzon marinus) and African Lungfish (Protopterus annectens): A Closer Look into the Role of the Non-gastric H+/K+-ATPase

Abstract

Fishes living in freshwater need to actively compensate for the diffusive loss of ions and osmotic gain of water. The gill is the primary organ of ion regulation and contains an array of ion transport proteins to help maintain homeostasis. Two of the more well studied ion pumps are the Na+/K+-ATPase (NKA) and vacuolar type proton ATPase (V-ATPase). This thesis focuses on another ion pump known as the non-gastric H+/K+-ATPase (ngHKA). The ngHKA (gene: atp12a) has not been found in any of the teleost fishes, indicating loss from that lineage. In contrast, there is confirmed expression in lamprey (Petromyzon marinus) and lungfish (Protopterus annectens) and thus these species were used to investigate the role of the ngHKA in fishes. It was hypothesized that the ngHKA plays a role in potassium and/or acid-base regulation in these fishes. The first objective was to develop an antibody as a tool to study the ngHKA. Next, was to characterize tissue expression patterns to infer functional significance. Finally, I wanted to confirm the pump’s functional significance by determining if potassium (fed-fasting) and acid-base challenges (3µEq/g wet mass H+ or HCO3- load) modulate the expression of the ngHKA. The pump was studied at multiple levels including; transcript expression using qPCR, protein expression using Western blotting, localization using immunohistochemistry, as well as potassium and proton ion flux measurements. Rubidium (Rb+) was used as a surrogate flux marker for potassium. The NKA and V-ATPase were also studied in an attempt to understand overall osmoregulation strategies. A rabbit polyclonal antibody (LF12Arb2) was successfully validated for the use in Western blotting and immunohistochemistry in both lamprey and lungfish. High expression of ngHKA was found in both gill and kidney, the major ion regulatory organs in fishes. Rubidium uptake was also successfully measured in both species. In lamprey, fed fish had significantly greater acid uptake and high Rb+ uptake rates against the predictions. Acid loaded lamprey had higher acid excretion rates compared to controls as predicted. There were no differences in ngHKA protein expression for any of the treatments and localization did not appear to change. Transcript (mRNA) levels did not change for any treatments in either lamprey or lungfish. In lungfish, the ngHKA and NKA protein expression decreased in omeprazole treated fish although with no corresponding changes in H+ or Rb+ flux rates. Apical staining did appear more pronounced in the acid loaded fish compared to controls, which should be investigated further. The results show no clear support for the hypothesis that the ngHKA plays a role in potassium and/or acid-base regulation. This was the first study to investigate the ngHKA in fishes. Future studies are suggested to use radioisotopes (86Rb) to advance the methods as well as perform immunohistochemistry quantification analysis