Effects of metabolic inhibition on cell volume of goldfish hepatocytes

4. ESPELT, M.V., PAFUNDO, D., MUT, P., KRUMSCHNABEL, G., AMODEO, G., P.J. SCHWARZBAUM

Buenos Aires

Simposio; Sattelite Symposium (XIV International Biophysics Congress): Volume regulation in animal and plant cells; 2002

In hepatocytes of most vertebrate species, blockage of oxidative phosphorylation leads to a cascade of events including depletion of ATP, membrane depolarization, intracellular acidification, a rise of cytosolic Ca2+, Mg2+ and Na+ and cell swelling. In hepatocytes of the goldfish, a facultative anaerobe vertebrate, these changes are slow to develop or do not occur at all. In anoxic hepatocytes from goldfish, metabolic adjustments that prolong cell survival include: 1) the reallocation of metabolic energy to essential functions, 2) the prevention of cytosolic Ca2+ increase by a net efflux of the cation; and 3) the blockage of acidification by the high intracellular buffer capacity (1). In order to properly predict the consequences of these changes, the associated osmotic disturbance has to be evaluated. Thus, goldfish hepatocytes were exposed to 2 mM CN and cell volume (Vc) was assessed. Parallel measurements of K+ transmembrane fluxes we carried out to discern whether the putative loss of the cation could be used to regulate cell volume under metabolic inhibition. In previous reports we have shown that the maintenance of intracellular K+ is a very sensitive indicator of both cell viability, although transitory loss of K+ can be used for volume regulatory responses. Changes in Vc were monitored by fluorescence microscopy using calcein, efflux and influx of K+ were assessed using the K+ analog 86Rb+. In anisotonic media, cells displayed a reversible osmometric change of Vc. In the presence of 2 mM CN-, a slight volume decrease was observed, whereas anoxic cells displayed a 50 % decrease of K+ influx and no change in K+ efflux. Using data from this and previous studies we predicted the changes in concentration of the main intracellular osmolytes during a 40 min exposure to CN- (in nmol osmolyte/106 cells): K+ -5.2; lactate –40.0; glucose -5.0; glycogenolysis-derived glycosyl units +72.1; ATP +1.5. An analysis of the relative increase or decrease of different organic and inorganic osmolytes under anoxia shows that accumulation of glycosyl units and production and transport of lactate –two processes which strongly depend on the rate of glycogenolysis-, can influence cell volume significantly.