Cellular and molecular basis of anoxia-tolerance and -intolerance in vertebrates. A comparative study using isolated goldfish and trout hepatocytes.

Cell physiological aspects anoxia-tolerance will by studied means of goldfish and trout hepatocytes as models of anoxia-tolerant and -intolerant cell-systems, respectively. In this context the following major aspects will be addressed: (1) What is the specific role of the maintenance of ion homeostasis during anoxia and by which mechanisms is this maintenance achieved? (2) Are oxygen-sensing mechanisms involved in the induction and/or the maintenance of a depressed metabolic state and what is the nature of these mechanisms? (3) Is glycolytic ATP production of a generally higher importance in anoxia-tolerant cells as compared to anoxia-intolerant cells and are there certain cellular functions directly dependent on ATP derived from glycolysis? The role of membrane function during anoxia shall be evaluated by creating, conditions of near complete arrest of ion cycling across the cell membrane and evaluating the effects of this condition on (a) cellular energy turnover, (b) intracellular pH and cell volume, and (c) on the expression of oxygen-regulated proteins. As to the mechanism responsible for the concerted shutdown of unidirectional ion fluxes the potential involvement of arachidonic acid and/or its derivatives will be studied. The existence of oxygen-sensitive proteins and their role in metabolic depression will be studied by comparing effects of true and chemical anoxia and of several effectors interfering with the function of oxygen-sensing proteins (Ni 2 , Co2 , carbon monoxide) on energy metabolism and on the hypothetical expression and depression of specific sets of proteins. The importance of glycolytic ATP production will be addressed by assessing the impact of glycolytic inhibition on (a) aerobic ATP-turnover, (b) K- and Ca2 - homeostasis, and (c ) protein synthesis. Furthermore, by challenging ion homeostasis in cells in the absence or presence of glycolytic inhibitors the tightness of coupling between membrane function and glycolytic ATP- provision shall be revealed.

For the cells of most vertebrates, a decrease in the availability of oxygen leads to energetic failure and ultimately cell death. During the last decades, innumerable studies have been conducted aimed at identifying the mechanisms underlying this fact and/or developing intervention strategies counteracting anoxia-induced cell damage. In the vast majority of these studies, cells from anoxia-sensitive organisms have been used as a model system. Only during the last few years, has a different approach to the question of anoxia-sensitivity been applied. Researchers were seeking an answer to the causes determining anoxia-sensitivity by exploiting the fact that among the many vertebrate species known there exist a few outstanding examples that are characterised by an unusual degree of anoxia-tolerance. Circumventing the often trial-and-error-like search for useful measures against anoxia-induced cell damage, this approach takes advantage of the fact that during evolution adequate strategies have already been established, the success of which is obvious from the mere existence of the species examined. The results obtained in these studies may differ from species to species and often lead to insights not directly transferable to humans. Nevertheless these studies have provided and will provide an invaluable basis for further research by both broadening our general understanding of anoxia-sensitivity and by more clearly defining the direction which future work on this subject should follow. In the present project the following major aspects have been addressed: (1) Is glycolytic ATP production of a generally higher importance in anoxia-tolerant cells as compared to anoxia-intolerant cells and are there certain cellular functions directly dependent on ATP derived from glycolysis? (2) What is the specific role of the maintenance of ion homeostasis during anoxia and by which mechanisms is this maintenance achieved? (3) Are oxygen-sensing mechanisms involved in the induction and/or the maintenance of a depressed metabolic state and what is the nature of these mechanisms? In order to discern, which properties are specific for anoxia tolerant cells and which are typical for fish cells, the most relevant experiments were also conducted with the anoxia-intolerant hepatocytes from trout. Regarding the first aspect, we could show that in hepatocytes from goldfish energy metabolism is to a large extent dependent on glycolytic flux as provider of both ATP and substrate, whereas in trout hepatocytes mitochondrial ATP production is sufficient to maintain energetic homeostasis. Furthermore, normoxic Na+ -pump activity was found to be partially dependent on glycolytic ATP in goldfish but not in trout cells. This coupling of glycolytic ATP production and Na+ -pump activity, in turn, may be regarded as the basis of the capability of the goldfish cells to preserve ion homeostasis under anoxia. Besides maintaining coupled K + fluxes and intracellular free Ca2+ levels, goldfish hepatocytes were able to maintain intracellular pH constant, whereas it was significantly decreased in the trout cells. In addition, the goldfish cells preserved cellular responsiveness to adrenergic stimulation, thereby ensuring glucose supply of tissues lacking ample glycogen reserves as present in the liver. Cellular sensing of oxygen lack appears to be mediated by oxygen-sensitive proteins, including hypoxia-inducible factor (HIF 1-alpha), as recently described for trout cells. Experimental evidence has been presented that oxygen radicals are involved in the transducing mechanism.