CHRONIC HYPERAMMONEMIA IMPAIRS THE GLUTAMATE-NITRIC OXIDE-CYCLIC GMP PATHWAY IN CEREBELLAR NEURONS IN CULTURE AND IN THE RAT IN-VIVO

The aim of this work was to assess whether ammonia concentrations simi lar to the increase found in the brain of hyperammonemic rats (100 mu M), impair N-methyl-D-aspartate (NMDA) receptor-mediated signal transd uction, We first measured glutamate neurotoxicity, which in these neur ons is mediated by activation of NMDA receptors, as an initial paramet er reflecting activation of NMDA receptor-mediated pathways. Long-term treatment of cultured neurons with ammonia prevents glutamate-induced neuronal death. The EC50 was 20 mu M, and at 100 mu M the protection was complete. The induction of the protective effect was not immediate , but took several hours. Treatment with 100 mu M ammonia did not prev ent a glutamate- or NMDA-induced rise of intracellular calcium, Ammoni a impaired the glutamate-nitric oxide-cGMP (3',5'-cyclic guanosine mon ophosphate) pathway in a dose- and time-dependent manner. Glutamate-in duced formation of cGMP was reduced by 42%, while activation of nitric oxide synthase was not affected. Ammonia reduced by 31% cGMP formatio n induced by S-nitroso-N-acetyl-penicillamine (SNAP), a NO-generating agent, confirming that the interference occurs at the level of guanyla te cyclase activation by nitric oxide. To assess whether chronic moder ate hyperammonemia in vivo also impairs the glutamate-nitric oxide-cGM P pathway, we determined by in vivo brain microdialysis in freely movi ng 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 a nimal in vivo also impairs the glutamate-nitric oxide-cGMP pathway. Im pairment of this pathway can contribute to the neurological alteration s found in hyperammonemia and hepatic encephalopathy.