Ammonia is a neurotoxin whose administration in large doses causes coma and death of the exposed animals, but whether and in what degree these whole body effects are related to the death of CNS cells is not known. Since the downstream effects of ammonia in cultured CNS cells appear to be partly mediated by overactivation of several putative signalling mechanisms characteristic for the apoptotic program, we speculated that ammonia neurotoxicity may be apoptogenic. In this study, C6 glioma cells grown in 2% serum were exposed to 5 mM or 10 mM NH(4)Cl (ammonia) for 96 h and tested for the appearance of apoptosis by (a) Hoechst staining, (b) TUNEL reaction and (c) DNA ladder, at different times of exposure. In cultures exposed to either 5 mM or 10 mM ammonia, about 10% of the cells were found to enter apoptosis at 48 h of exposure, and the number of apoptotic cells rose to 30% at 72 h, and to 50% at 96 h of exposure, respectively. The first transduction signal purportedly involved in apoptosis, activation of PKCalphabeta, was transient and appeared already after 3-6 h of treatment. Coincident with pronounced manifestation of apoptosis (at 72 h and even more at 96 h of exposure) was an increased transfer of the transcription factor NFkappaB from cytoplasmto nucleus as revealed by EMSA assay. The number of cells affected by ammonia-induced apoptosis was markedly reduced by incubation with a NOS inhibitor, L-NAME at 100 microM concentration. The results indicate that ammonia-induced apoptosis is a result of a complex interplay of at least three signalling molecules: NO, PKC and the transcription factor NFkappaB, with NFkappaB being possibly involved in the induction of iNOS and generation of toxic levels of NO in the cells.