EFFECT OF AMMONIUM-CHLORIDE ON ENERGY-METABOLISM OF ASTROCYTES AND C6-GLIOMA CELLS IN-VITRO

Increased ammonia has been considered a key factor in the pathogenesis of hepatic encephalopathy. The high concentration of ammonia interfer es with oxidative metabolism in the brain through an inhibitory effect on the tricarboxylic acid cycle (TCA). Inhibition of the TCA cycle ma y result in depletion of ATP. Due to the involvement of astrocytes in brain detoxification of ammonia, these cells are good candidates for s tudying ammonia's effect on energy stores in the brain. C6-glioma cell s, which have altered glycolytic rates, may show greater sensitivity t o the toxicity of ammonium chloride than astrocytes. To study the effe ct of ammonium chloride on energy storage of both astrocytes and C6-gl ioma, we observed the acute and chronic effects of NH4Cl (7.5 or 15 mM ) on the metabolism of isolated astrocytes and C6-glioma cells. Primar y astrocytes were isolated from the cerebral hemispheres of 1-2 day ol d Sprague-Dawley rats, and C6-glioma cells were purchased from the Ame rican Type Culture Collection (ATCC). Following treatment of the cells with ammonia, glucose, lactate, glutamate, ATP, and PCr were assayed. Our data showed that at 15 min following treatment with NH4Cl, there were no significant differences in the concentration of metabolites me asured in astrocytes. However, following 15 min of treatment with NH4C l, the concentration of some metabolites, for example, ATP and lactate , changed significantly in C6-glioma cells. We have shown that 24 h of treatment was sufficient time to see significant biochemical changes but not morphological changes in either cell type. Simultaneous bioche mical and. morphological changes were observed 48 h following treatmen t in C6-glioma cells and at 9-10 days following treatment in primary a strocytes. In primary astrocytes at 24 h following treatment, glucose utilization increased. This high utilization of glucose was in accorda nce with the increase in lactate and glutamate production and the decr ease in ATP and PCr formation. In C6-glioma cells the utilization of g lucose increased but this high utilization of glucose was consistent w ith a significant decrease in the concentration of lactate, glutamate and ATP.