GABAC receptor agonist suppressed ammonia‐induced apoptosis in cultured rat hippocampal neurons by restoring phosphorylated BAD level

Ammonia‐induced apoptosis and its prevention by GABAC 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 anti‐neurofilament antibody. A GABAC receptor agonist, cis‐4‐aminocrotonic acid (CACA, 200 µm), but not GABAA and GABAB receptor agonists, muscimol (10 µm) and baclofen (50 µm), respectively, inhibited the ammonia (2 mm)‐induced apoptosis, and this inhibition was abolished by a GABAC receptor antagonist (1,2,5,6‐tetrahydropyridin‐4‐yl)methylphosphinic acid (TPMPA, 15 µm). Expression of all three GABAC 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 µm) and KT5720 (1 µm), suggesting possible involvement of an upstream pro‐apoptotic protein, BAD. Levels of phospho‐BAD (Ser112 and Ser155) were decreased by the ammonia‐treatment and restored by coadministration of CACA. These findings suggest that GABAC receptor stimulation protects hippocampal pyramidal neurons from ammonia‐induced apoptosis by restoring Ser112‐ and Ser155‐phospho‐BAD levels.