Ammonium injection induces an N-methyl-D-aspartate receptor-mediated proteolysis of the microtubule-associated protein MAP-2.
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 approximately 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 Ca(2+)-dependent protease that selectively degrades MAP-2. We show that there is a cytosolic Ca(2+)-dependent protease that degrades MAP-2, but not 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Felipo
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Instituto de Investigaciones Citológicas, Fundación Valenciana de Investigaciones Biomédicas, Spain.
Grau
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Miñana
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MD
Grisolía
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D000641:Ammonia / Q000494:pharmacology*; D000818:Animals; D002118:Calcium / Q000502:physiology; D002154:Calpain / Q000037:antagonists & inhibitors; D002331:Carnitine / Q000494:pharmacology; D016291:Dizocilpine Maleate / Q000494:pharmacology; D007274:Injections, Intraperitoneal; D008297:Male; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D016194:Receptors, N-Methyl-D-Aspartate / Q000502:physiology*
D008869:Microtubule-Associated Proteins; D011480:Protease Inhibitors; D016194:Receptors, N-Methyl-D-Aspartate; D016291:Dizocilpine Maleate; D000641:Ammonia; D010447:Peptide Hydrolases; D002154:Calpain; D002331:Carnitine; D002118:Calcium