Teleost fish are very sensitive to changes that occur in their aquatic environment, including those that influence blood acid-base status. The intracellular pH (pHi) of the liver is strongly influenced by the pH of circulating blood and must be able to regulate any changes that occur. The main goal of this thesis was to examine the mechanisms of pHi regulation in hepatocytes (liver cells) isolated from different fish species. Isolated hepatocytes were validated as an appropriate cell model system by examining intracellular and plasma ion levels in three teleost species, rainbow trout, Oncorhynchus mykiss (Walbaum), black bullhead, Ameiurus melas (Rafinesque) and American eel, Anguilla rostrata (Lesueur). Although the electrochemical gradients for Na + and K+ were different in isolated cells as compared to the intact tissue, these gradients were directionally correct. The mechanism of intracellular pH (pHi) regulation was studied in trout, bullhead and eel hepatocytes using the pH-sensitive fluorescent dye BCECF. In the trout and bullhead, recovery to an acid load occurred principally by way of Na+-H+ exchange. In trout hepatocytes, an electrogenic Na+-HCO3- cotransporter also plays a significant role in the maintenance of steady-state pHi and recovery from intracellular acidosis. In eel, recovery to an acid load occurred by a Cl--dependent mechanism. Recovery to a base load in the trout was diminished by blocking the Na+-H + exchanger. Partial sequences of the Na+-HCO3- (NBC) gene were obtained independently from trout liver and gill/kidney cDNA libraries. The fish NBC sequences were identical and showed high homology with mammalian and amphibian sequences. The most important differences between the fish and these other sequences occurred in the large extracellular loop as well as in the DIDS binding motif. Hypercapnia exposure resulting in decreases in blood pH significantly decreases hepatocyte pHi. Chronic in vivo or in vitro hypercapnia did not enhance the ability of hepatocytes to recover from subsequent intracellular acidoses. In summary, hepatocytes of all three teleost species studied appear to have the ability to regulate pHi, though they do not exploit identical mechanisms. In addition to the role of a Na+-H+ exchanger and Cl--HCO3- exchanger, a Na+-HCO3- cotransporter may contribute significantly to hepatocyte pHi regulation. The primary molecular data obtained in this thesis may allow for a more complete and in depth determination of a role of the NBC in regulatory mechanisms in teleost fish.
| Identifer | oai:union.ndltd.org:uottawa.ca/oai:ruor.uottawa.ca:10393/9437 |
| Date | January 2001 |
| Creators | Furimsky, Marosh. |
| Contributors | Moon, Thomas W., |
| Publisher | University of Ottawa (Canada) |
| Source Sets | Université d’Ottawa |
| Detected Language | English |
| Type | Thesis |
| Format | 182 p. |
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