Chronic Exposure to Ammonia Alters Pathways Modulating Phosphorylation of Microtubule-Associated Protein 2 in Cerebellar Neurons in Culture Rosana Sa ´ez, Marta Llansola, and Vicente Felipo Laboratory of Neurobiology, Instituto de Investigaciones Citologicas, Fundacio ´n Valenciana de Investigaciones Biome ´dicas, Valencia, Spain Abstract: Hyperammonemia is considered the main cause for the neurological alterations found in hepatic failure. However, the mechanisms by which high ammo- nia levels impair cerebral function are not well under- stood. It has been shown that chronic hyperammonemia impairs signal transduction pathways associated with NMDA receptors and also alters phosphorylation of some neuronal proteins. The aim of the present work was to analyze the effects of chronic exposure to ammonia on phosphorylation of microtubule-associated protein 2 (MAP-2) in intact neurons in culture and to assess whether modulation of MAP-2 phosphorylation by gluta- mate receptor-associated transduction pathways is al- tered in neurons chronically exposed to ammonia. It is shown that chronic exposure to ammonia increases basal phosphorylation of MAP-2 by 70%. This effect seems to be due to a decreased tonic activation of NMDA re- ceptors and of calcineurin. Chronic exposure to ammonia also alters the modulation of MAP-2 phosphorylation by NMDA receptors and metabotropic glutamate receptors. In neurons exposed to ammonia, treatment with NMDA for 30 min induced a significant decrease in phosphory- lation of MAP-2. Activation of metabotropic glutamate receptors with (1S,3R)-1-aminocyclopentane-1,3-dicar- boxylic acid significantly increased phosphorylation of MAP-2 in control neurons, whereas in neurons exposed to ammonia the response was the opposite, with 1-ami- nocyclopentane-1,3-dicarboxylic acid inducing a de- phosphorylation of MAP-2. These results indicate that ammonia alters significantly signal transduction path- ways associated with different types of glutamate recep- tors. This would lead therefore to significant alterations in glutamatergic neurotransmission, which would contrib- ute to the neurological alterations found in hyperam- monemia and in hepatic encephalopathy. Key Words: Hepatic encephalopathy—NMDA receptors—Metabo- tropic glutamate receptors—Signal transduction— Microtubules—Hyperammonemia. J. Neurochem. 73, 2555–2562 (1999). Hepatic encephalopathy is a neuropsychiatric syn- drome that occurs in patients with liver failure. Hepatic encephalopathy affects intellectual function and person- ality and can lead to seizures, convulsions, coma, and death. The neurobiological bases of hepatic encephalop- athy are not clearly understood. Ammonia is considered the main entity responsible for the neurological alter- ations found in hepatic encephalopathy: Increased am- monia levels lead to alterations in the function of the CNS and can lead to coma and death. However, the molecular bases for ammonia-induced alterations in ce- rebral function remain unclear. It has been shown that chronic exposure to ammonia impairs signal transduction pathways associated with NMDA receptors (Marcaida et al., 1995; Min ˜ana et al., 1995; Hermenegildo et al., 1998). We have also previously found indirect evidence that hyperammonemia leads to alterations in the phos- phorylation of neuronal proteins such as microtubule- associated protein 2 (MAP-2) (Felipo et al., 1993a; Grau et al., 1996), Na + /K + -ATPase (Kosenko et al., 1994), and the NMDA receptor (Marcaida et al., 1995). These neuronal proteins play important roles in neurotransmis- sion and are modulated by phosphorylation. An ammo- nia-induced alteration of the phosphorylation of these proteins could therefore play a role in the mediation of altered neurotransmission in hyperammonemia, leading to neurological alterations. Phosphorylation of MAP-2 is modulated by signal transduction pathways associated with different types of glutamate receptors (Halpain and Greengard, 1990; Quinlan and Halpain, 1996a,b). Activation of either NMDA receptors or metabotropic glutamate receptors (mGluRs) modulates phosphorylation of MAP-2 through signal transduction pathways that have not been com- pletely identified. Received May 18, 1999; revised manuscript received July 29, 1999; accepted August 2, 1999. Address correspondence and reprint requests to Dr. V. Felipo at Laboratory of Neurobiology, Instituto de Investigaciones Citolo ´gicas, Fundacio ´n Valenciana de Investigaciones Biome ´dicas, Amadeo de Saboya, 4, 46010 Valencia, Spain. Abbreviations used: t-ACPD, (1S,3R)-1-aminocyclopentane-1,3-di- carboxylic acid; MAP-2, microtubule-associated protein 2; mGluR, metabotropic glutamate receptor. 2555 Journal of Neurochemistry Lippincott Williams & Wilkins, Inc., Philadelphia © 1999 International Society for Neurochemistry