Eugenio Grau

Brain ATP Depletion Induced by Acute Ammonia Intoxication in Rats Is Mediated by Activation of the NMDA Receptor and Na+, K+‐ATPase

Abstract: Injection of large doses of ammonia into rats leads to depletion of brain ATP. However, the molecular mechanism leading to ATP depletion is not clear. The aim of the present work was to assess whether ammonium‐induced depletion of ATP is mediated by activation of the NMDA receptor. It is shown that injection of MK‐801, an antagonist of the NMDA receptor, prevented ammonia‐induced ATP depletion but did not prevent changes in glutamine, glutamate, glycogen, glucose, and ketone bodies. Ammonia injection increased Na+,K+‐ATPase activity by 76%. This increase was also prevented by previous injection of MK‐801. The molecular mechanism leading to activation of the ATPase was further studied. Na+,K+‐ATPase activity in samples from ammonia‐injected rats was normalized by “in vitro” incubation with phorbol 12‐myristate 13‐acetate, an activator of protein kinase C. The results obtained suggest that ammonia‐induced ATP depletion is mediated by activation of the NMDA receptor, which results in decreased protein kinase C‐mediated phosphorylation of Na+,K+‐ATPase and, therefore, increased activity of the ATPase and increased consumption of ATP.

Nitroarginine, an Inhibitor of Nitric Oxide Synthetase, Attenuates Ammonia Toxicity and Ammonia-Induced Alterations in Brain Metabolism

We have proposed that acute ammonia toxicity is mediated by activation of the N-methyl-D-aspartate type of glutamate receptors. MK-801, a selective antagonist of these receptors, prevents death of animals induced by acute ammonia intoxication as well as ammonia-induced depletion of ATP. It seems therefore that, following activation of the N-methyl-D-aspartate receptors, the subsequent events in ammonia toxicity should be similar to those involved in glutamate neurotoxicity. As it has been shown that inhibitors of nitric oxide synthetase such as nitroargnine prevent glutamate toxicity, we have tested whether nitroarginine prevents ammonia toxicity and ammonia-induced alterations in brain energy and ammonia metabolites. It is shown that nitroarginine prevents partially (≈50%), but significantly death of mice induced by acute ammonia intoxication. Nitroarginine also prevents partially ammonia-induced depletion of brain ATP. It also prevents completely the rise in glucose and pyruvate and partially that in lactate. Injection of nitroarginine alone, in the absence of ammonia, induces a remarkable accumulation of glutamine and a decrease in glutamate. The results reported indicate that nitroarginine attenuates acute ammonia toxicity and ammonia-induced alterations in brain energy metabolites. The effects of MK-801 and of nitroarginine are different, suggesting that ammonia can induce nitric oxide synthetase by mechanisms other than activation of N-methyl-D-aspartate receptors.

Ammonium Injection Induces an N-Methyl-d-Aspartate Receptor-Mediated Proteolysis of the Microtubule-Associated Protein MAP-2

Abstract: We have shown previously that chronic hyperammonemia increases, in brain, the polymerization of microtubules that is regulated mainly by the level and state of phosphorylation of microtubule‐associated protein 2 (MAP‐2). Activation of the N‐methyl‐d‐aspartate (NMDA) receptor dephosphorylates MAP‐2. Because we have found that acute ammonia toxicity is mediated by the NMDA receptor, we have tested the effect of high ammonia levels on MAP‐2 in brain. Microtubules isolated from rats injected intraperitoneally with 6 mmol/kg ammonium acetate showed a marked decrease of MAP‐2. Also, the amount of MAP‐2 in brain homogenates, determined by immunoblotting. was markedly reduced, presumably by proteolysis. The content of MAP‐2 was decreased by ∼75% 1‐2 h after ammonium injection and returned to normal values after 4 h. Proteolysis of MAP‐2 was prevented completely by injection of 2 mg/kg MK‐801, a specific antagonist of the NMDA receptor, suggesting that proteolysis is mediated by activation of this receptor. l‐Carnitine, which protects rats against ammonia toxicity, also prevented MAP‐2 degradation. Because activation of the NMDA receptor increases [Ca2+]i, we determined whether rat brain contains a Ca2+‐dependent protease that selectively degrades MAP‐2. We show that there is a cytosolic Ca2+‐dependent protease that degrades MAP‐2, but no other brain proteins. The protease has been identified tentatively as calpain I, for it is inhibited by a specific inhibitor of this protease. Our results suggest that ammonium injection activates the NMDA receptor, leading to an increase in [Ca2+]i, which activates calpain I. This, in turn, selectively degrades MAP‐2. Possible implications in chronic hyperammonemic states and in the mechanism of ammonia toxicity are discussed.

Ammonium ingestion prevents depletion of hepatic energy metabolites induced by acute ammonium intoxication

Ingestion of an ammonium containing diet produces hyperammonemia and protects rats against acute ammonium intoxication. Acute ammonium toxicity has been attributed to the depletion of energy metabolite intermediates. We show here that hyperammonemia affords considerable protection against depletion of hepatic energy metabolites evoked by ammonium acetate injection. In control rats there were marked decreases in the content of acetoacetate, beta-hydroxybutyrate, ATP, 2-oxoglutarate, lactate, and pyruvate while phosphoenolpyruvate increased markedly. In hyperammonemic rats beta-hydroxybutyrate, ATP, 2-oxoglutarate, and lactate were not significantly affected while pyruvate increased markedly and phosphoenolpyruvate slightly. These results suggest that in controls the activity of pyruvate kinase is inhibited after ammonium injection while in hyperammonemic rats it is not inhibited. The content of alanine (an inhibitor of pyruvate kinase) reached 2.8 mumol/g in controls and 1.6 mumol/g in hyperammonemic rats, 15 min after ammonium injection. This could explain the different effects of ammonium injection on control and hyperammonemic rats.

Effects of Hyperammonemia on Brain Protein Kinase C Substrates

Ammonia is a product of the degradation of proteins and of other compounds ; however, when it is in excess, ammonia is a toxic compound . A fiveto ten-fold increase in blood ammonia levels leads to alterations in the function of the central nervous system, and can lead to coma and death . To prevent these toxic effects, ureotelic animals have developed the urea cycle, which is mainly located in liver, and eliminates ammonia by incorporating it into urea, which is eliminated in urine . This maintains safe levels of ammonia in blood and tissues. However, when this process fails due to a congenital defect in the urea cycle enzymes or by impairment of liver function, the levels of ammonia in blood rise and can lead to altered brain function. This syndrome, known as hepatic encephalopathy, can lead to coma and death. Ammonia interferes with neurotransmission and with electrophysiological processes (Fan et al., 1990; Szerb and Butterworth, 1992 ; Raabe and Lin, 1984 ; Raabe, 1992 and 1994, Butterworth, 1994) . There are a number of human illnesses that are associated with increased levels of ammonia in blood, including liver cirrhosis, fulminant hepatic failure and congenital defects of urea cycle enzymes . Independently of its origin, in these situations the levels of ammonia in blood increase 5 to 10-fold, leading to hepatic encephalopathy, and high mortality . In fact, hepatic encephalopathy is one of the main causes of death in occidental countries . In Spain 12,000 people die every year for this reason, which represents about

Activation of NMDA Receptor Mediates the Toxicity of Ammonia and the Effects of Ammonia on the Microtubule-Associated Protein MAP-2

Hepatic encephalopathy is one of the main causes of death in western countries. In Spain, nearly 12,000 people die every year by liver cirrhosis (-8,000) or other hepatic failures; this represents approximately 4% of the total number of deaths. In spite of much work, the actual cause of hepatic coma and death remains unclear.

Protein kinase C isoforms and cell proliferation in neuroblastoma cells

The expression of protein kinase C isoforms in the neuroblastoma cell line Neuro 2a has been studied. It is shown that Neuro 2a cells express alpha, delta, epsilon and zeta PKCs. Inhibition of cell proliferation by using protein kinase C inhibitors (H7 or calphostin C) or medium without glutamine affects markedly the pattern of PKC isoforms. All treatments reduced significantly (50-70%) the content of PKC alpha. None of the treatments altered PKC zeta or epsilon. The content of PKC delta was increased (88-120%) in cells treated with PKC inhibitors but was slightly reduced in cells incubated in medium without glutamine. However, none of the treatments affected the content of the corresponding mRNAs. Long-term treatment of synchronized cells with the phorbol ester PMA depletes PKC alpha but not PKC delta or zeta and only partially PKC epsilon. This treatment with PMA did not affect DNA synthesis, indicating that PKC alpha does not play a significant role in the control of proliferation of these cells.

Control of Urea Synthesis and Ammonia Detoxification

Ammonia toxicity was first reported by Pavlov and coworkers a century ago (1). They excluded the liver from the circulation and found that when dogs treated in this way were fed meat, they developed hyperammonemia, which was associated with coma and led to the death of the animal.

The susceptibility of MAP-2 to proteolytic degradation increases when bound to tubulin.

During experiments studying dietary effects on phosphorylation/dephosphorylation of MAP-2 we found that incubation of microtubules with alkaline phosphatase resulted in extensive proteolysis of MAP-2 but not of tubulin or Tau proteins. In the absence of tubulin, when microtubule-associated proteins (MAPs) were incubated with alkaline phosphatase, MAP-2 was not proteolyzed. This suggests that binding to tubulin induces a conformational change in MAP-2 which makes it more susceptible to proteolysis. The proteolysis of MAP-2 by alkaline phosphatase was prevented by inhibitors of serine proteases, suggesting that the commercial preparation of the enzyme is contaminated by a serine protease and/or that the enzyme also has a weaker proteolytic activity. In addition, selective proteolysis of MAP-2 can be obtained with the metalloprotease collagenase. Brain homogenates are shown to contain a Ca2+-dependent protease which selectively degrades MAP-2 bound to tubulin. These results suggest that selective proteolysis of tubulin-bound MAP-2 could play a role in the regulation of microtubule dynamics in response to extracellular signals.