Ammonia effects on pyruvate/lactate production in astrocytes—Interaction with glutamate

Abstract

Ammonia exerts a multitude of metabolic and non-metabolic effects on brain tissue. In the present communication we have investigated its effect on lactate production rates, pyruvate production rates and pyruvate/lactate ratios in mouse cerebrocortical astrocytes and neurons in primary cultures. No effects were found in neurons. All three parameters were affected by ammonia in astrocytes, but less potently and to a smaller degree in cells that had been treated with dibutyryl cyclic AMP (morphologically differentiated cells) than in untreated cells (morphologically undifferentiated cells). In the differentiated cells ammonia had virtually no effect up to a concentration of 1.0 mM, but at 3.0 mM it increased lactate production and decreased pyruvate/lactate ratio significantly. In the undifferentiated cells ammonia greatly increased lactate accumulation (by 80% at 3.0 mM) and it inhibited pyruvate accumulation (by 40% at 3.0 mM). It thereby reduced the pyruvate/lactate ratio progressively within the entire range 0.1–3.0 mM ammonia. In support of the hypothesis that the ammonia-induced reduction of pyruvate/lactate ratio is secondary to depletion of cellular glutamate by formation of glutamine (and glutathione) and a resulting interruption of the malate–aspartate shuttle (MAS), the addition of glutamate to the incubation medium significantly diminished the ammonia-induced reduction of pyruvate/lactate ratio, whereas it had no effect on the increased lactate production. It is discussed that MAS interruption may have additional consequences in astrocytes.

Introduction

Glucose utilization is increased in brains of acutely hyperammonemic animals (Schenker et al., 1967, Hindfelt and Siesjö, 1971, Hawkins et al., 1973, James et al., 1974), an ammonia-induced stimulation of glycolysis has been demonstrated in brain slices (Ashford and Dixon, 1935, McKhann and Tower, 1961), and ammonia activates brain phosphofructokinase, a rate-limiting enzyme in glycolysis (Lowry and Passonneau, 1966, Sugden and Newsholme, 1975). The stimulation of phosphofructokinase has been confirmed by Ratnakumari and Murthy (1993), who observed elevated activities of several glycolytic enzymes (phosphofructokinase, aldolase, glyceraldehyde-3-phosphate dehydrogenase, enolase and pyruvate kinase) in cerebral cortex, cerebellum and brainstem of rats treated with ammonium acetate.

The increased glycolysis observed in brains of hyperammonemic animals is not mirrored by a similar increase in pyruvate concentration, since the pyruvate/lactate ratio is reduced, reflecting a decrease in cytosolic [NAD⁺/NADH] ratio (Hindfelt and Siesjö, 1971, Gjedde et al., 1978). Hindfelt et al. (1977) suggested that the change in cytosolic redox state together with an oppositely directed change in mitochondrial [NAD⁺/NADH] ratio may be due to interference with malate–aspartate shuttle (MAS) activity, which is essential for transporting reduced equivalents generated during glycolysis in the cytosol to the mitochondria (Fig. 1). Since glutamate is an essential component of MAS, they further proposed that such an inhibition of MAS could be due to a reduction of the glutamate concentration, brought about by an enhanced glutamine formation for ammonia detoxification. When MAS is inhibited, cytosolic NADH generated during glycolysis cannot be re-oxidized by reduction of oxaloacetate to malate (Fig. 1), but must instead react with pyruvate to form lactate. Glutamine synthetase in brain is confined to astrocytes (Norenberg and Martinez-Hernandez, 1977, Derouiche, 2004), and there is consensus that astrocytes constitute a major target for ammonia toxicity in brain (Norenberg, 1998, Rama Rao and Norenberg, 2001, Bélanger et al., 2004).

In the present communication we have examined the effects of acute exposure to ammonia on lactate production, pyruvate production and pyruvate/lactate ratio in primary cultures of mouse cerebrocortical interneurons (Hertz et al., 1985), a GABAergic preparation, and of cortical astrocytes, a preparation displaying a multitude of metabolic characteristics of mature astrocytes in brain (Hertz et al., 1998, Hertz et al., 2000). Moreover, it was investigated whether the addition of glutamate to the incubation medium altered either rates of lactate production or pyruvate/lactate ratios. Some of the findings have been reported in preliminary form (Hertz et al., 2000).