Chronic hyperammonemia impairs the glutamate–nitric oxide–cyclic GMP pathway in cerebellar neurons in culture and in the rat in vivo

Abstract

The aim of this work was to assess whether ammonia concentrations similar to the increase found in the brain of hyperammonemic rats (100 microM), impair N-methyl-D-aspartate (NMDA) receptor-mediated signal transduction. We first measured glutamate neurotoxicity, which in these neurons is mediated by activation of NMDA receptors, as an initial parameter reflecting activation of NMDA receptor-mediated pathways. Long-term treatment of cultured neurons with ammonia prevents glutamate-induced neuronal death. The EC50 was 20 microM, and at 100 microM the protection was complete. The induction of the protective effect was not immediate, but took several hours. Treatment with 100 microM ammonia did not prevent a glutamate- or NMDA-induced rise of intracellular calcium. Ammonia impaired the glutamate-nitric oxide-cGMP (3',5'-cyclic guanosine monophosphate) pathway in a dose- and time-dependent manner. Glutamate-induced formation of cGMP was reduced by 42%, while activation of nitric oxide synthase was not affected. Ammonia reduced by 31% cGMP formation induced by S-nitroso-N-acetyl-penicillamine (SNAP), a NO-generating agent, confirming that the interference occurs at the level of guanylate cyclase activation by nitric oxide. To assess whether chronic moderate hyperammonemia in vivo also impairs the glutamate-nitric oxide-cGMP pathway, we determined by in vivo brain microdialysis in freely moving rats the formation of cGMP induced by NMDA. In hyperammonemic rats, the formation of cGMP induced by NMDA and SNAP was reduced by ca. 60 and 41%, respectively, indicating that chronic hyperammonemia in the animal in vivo also impairs the glutamate-nitric oxide-cGMP pathway. Impairment of this pathway can contribute to the neurological alterations found in hyperammonemia and hepatic encephalopathy.

Oral administration of sildenafil restores learning ability in rats with hyperammonemia and with portacaval shunts

“…8 Moreover, we show herein that chronic treatment of these rats with sildenafil also restores the function of the pathway, extracellular cGMP, and learning ability.…”

“…7,8 This modulation is altered in patients who have died of hepatic encephalopathy, as it is in rats with portacaval anastomosis. 10 Therefore, the function of the glutamate-NO-cGMP pathway also should be altered in the brains of these patients as in rats with portacaval anastomosis and also should be responsible for the impairment of some intellectual functions.…”

“…8 Animals were allowed to recover for 24 to 48 hours in the microdialysis bowl of the system for freely moving animals (BAS BeeKeeper; Bioanalytical System, Lafayette, IN), with free access to food and water.…”

“…7,8 Chronic hyperammonemia also impairs the ability of rats to learn a conditional discrimination task. 9 The step of the glutamate-NO-cGMP pathway altered in rat models of hyperammonemia and hepatic encephalopathy is the activation of soluble guanylate cyclase by NO, 8,10 which is also altered in brains of patients who died with hepatic encephalopathy.…”

“…7,8 Chronic hyperammonemia also impairs the ability of rats to learn a conditional discrimination task. 9 The step of the glutamate-NO-cGMP pathway altered in rat models of hyperammonemia and hepatic encephalopathy is the activation of soluble guanylate cyclase by NO, 8,10 which is also altered in brains of patients who died with hepatic encephalopathy. 10 We hypothesized that the alterations in the function of the glutamate-NO-cGMP pathway in the brain in hyperammonemia and liver disease may be responsible for the impairment in learning ability and intellectual function and that pharmacological modulation of the pathway may restore learning ability in hyperammonemia and hepatic encephalopathy.…”