Ammonia toxicity under hyponatremic conditions in astrocytes: De novo synthesis of amino acids for the osmoregulatory response

Abstract

This study investigates how the metabolic activity and de novo synthesis of amino acids from glucose correlate with changes in intracellular organic osmolytes involved in astrocytic volume regulation during hyperammonemia and hyponatremia. Multinuclear ( 1H-, 31P-, 13C-) NMR spectra were recorded to quantify water-soluble metabolites, the cellular energy state, as well as the incorporation of [1- 13C]glucose into amino acids of primary astrocyte cultures. Myo-inositol levels were strongly decreased already at 3 h after treatment with NH 4Cl; other intracellular osmolytes, such as hypotaurine and choline-containing compounds were also decreased, along with a concomitant increase of both the total concentration and the amount of newly synthesized glutamine, alanine, and glutathione. During ammonia stress, the decrease of organic osmolytes compensated in part for increased intracellular osmolarity caused by amino acid synthesis. Hypotonic conditions alone also lowered the content of organic osmolytes including cellular amino acids, but much less than in hyperammonemia. This was due to impaired mitochondrial metabolic activity via the Krebs cycle, which also enhanced ammonia-induced ATP decrease. However, the changes in the sum of organic osmolytes were not significantly different after ammonia-treatment in hypoosmotic compared to anisoosmotic media, suggesting that the decrease of cellular organic osmolytes may not adequately compensate for the increased intracellular osmolarity caused by amino acids under hyponatremia. Therefore, the ammonia-induced release of osmolytes is an early process in response to increased intracellular osmolarity evoked by increased glutamine and alanine as a consequence of stimulated metabolic activity. The imperfect correlation of changes in astrocytic glutamine, other organic osmolytes, and the cellular energy state under hyperammonemic stress in isoosmotic and hypoosmotic media, however, point to additional mechanisms contributing to astrocyte dysfunction in hyperammonemic states, which are independent from glutamine formation.