UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Anoxia tolerant hepatocytes : a model system for the study of metabolic and channel arrest Buck, Leslie T.


Although there are a large number of studies on the adaptive mechanisms by which organisms tolerate and survive hypoxic or anoxic periods, this literature consists of experimental information gathered from a heterogeneous assemblage of species at the whole-animal and tissue level of organization, making a comprehensive understanding of the intracellular anoxic defense mechanisms difficult to obtain. A cell level system that responses to anoxia in a fashion similar to that of the anoxia-tolerant animal from which it was obtained would be invaluable. Described here is a method for the isolation of anoxia tolerant hepatocytes from the freshwater turtle Chrysemys picta bellii. Freshly isolated hepatocytes were determined to be viable based on trypan blue exclusion, LDH leakage, gluconeogenic capacity from 14C-lactate and responsiveness to adrenalin, glucagon, and insulin, and maintenance of cellular [adenylate]. During 10 h ofanoxic incubation cell staining, LDH leakage, and cellular [ATP] was unchanging, but the rate of ATP turnover decreased by 90%. The microcalorimetrically measured heat flux from hepatocytes in suspension (25°C) decreased by 76% in response to anoxia. To account for the difference between the two measurements of metabolic rate, the heat flux from known anaerobic end products was calculated (using caloric equivalents). Two dominant pathways, known to be functional (glycogen fermentation to lactate and the breakdown of glycogen to free glucose), accounted for only 42% of the anoxic heat flux, resulting in an "exothermic gap" (58%). This differs from the normoxically incubated hepatocytes where the indirect calorimetric measurement of heat flux (hepatocyte 02 consumption) could fully account for the calorimetrically measured heat flux. When normoxic hepatocytes were inhibited with cyanide a rapid monophasic suppression in heat flux was observed, suggesting that there is no short-term Pasteur Effect in these cells. Since [ATP] was unchanging in the presence of a 10 fold decrease in ATP turnover, a concomitant decrease in ATP utilization seemed essential. In view of the large fraction of total cellular energy metabolism used to support ion gradients the activity of the plasma membrane Na +/K + ATPase and membrane potential was measured in response to anoxia. From normoxic hepatocyte suspensions theouabain inhibitable 86Rb+ uptake was determined and found to comprise 28% of the total normoxic cellular ATP turnover. In response to anoxic incubation the activity of the pump decreased by 75%; however, this comprised 74% of the total anoxic ATP turnover. Plasma membrane potential was measured during anoxia, using the distribution of 36CI-, and was not significantly different from the normoxic measurement. Since the plasma membrane potential was maintained during anoxia, and since the activity of the Na +/K + ATPase decreased, the flux of ions across the plasma membrane must also have decreased (a low permeability state termed "channel arrest"). Taken together, an anoxia tolerant hepatocyte preparation has been developed that undergoes a 10 fold reduction in ATP turnover in response to anoxia (metabolic arrest). Furthermore, a 4 fold decrease in the rate of ATP utilization by Na +/K + ATPase, and the lack of a change in membrane potential, in response to anoxia suggests that mechanisms exist that achieve a functional decrease in ATP utilization and membrane permeability (channel arrest). Within a cellular system such as this the more complex regulatory mechanisms involved in a large coordinated reduction in ATP turnover rates can be probed during normoxic -anoxic transitions.

Item Media

Item Citations and Data


For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.