Ammonia-induced apoptosis and its prevention by GABA(c) receptor stimulation were examined using primary cultured rat hippocampal neurons. Ammonia (0.5-5 mm NH4Cl) dose-dependently induced apoptosis in pyramidal cell-like neurons as assayed by double staining with Hoechst 33258 and antineurofilament antibody. A GABA(c) receptor agonist, cis-4-aminocrotonic acid (CACA, 200 lion), but not GABA(A) and GABA(B) receptor agonists, muscimol (10 muM) and baclofen (50 muM), respectively, inhibited the ammonia (2 mm)-induced apoptosis, and this inhibition was abolished by a GABA(c) receptor antagonist (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA, 15 lion). Expression of all three GABA(c) receptor subunits was demonstrated in the cultured neurons by RT-PCR. The ammonia-treatment also activated caspases-3 and -9 as observed in immunocytochemistry for PARP p85 and western blot. Such activation of the caspases was again inhibited by CACA in a TPMPA-sensitive manner. The anti-apoptotic effect of CACA was blocked by inhibitors for MAP kinase kinase and cAMP-dependent protein kinase, PD98059 (20 muM) and KT5720 (1 muM), suggesting possible involvement of an upstream pro-apoptotic protein, BAD. Levels of phospho-BAD (Ser(112) and Ser(155)) were decreased by the ammonia-treatment and restored by coadministration of CACA. These findings suggest that GABA(c) receptor stimulation protects hippocampal pyramidal neurons from ammonia-induced apoptosis by restoring Ser(112)- and Ser(155)-phospho-BAD levels.
