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 microm), but not GABAA and GABAB receptor agonists, muscimol (10 micro m) and baclofen (50 microm), 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 microm). 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 microm) and KT5720 (1 microm), 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.
Yang
Li
L
Department of Pharmacology, Kansai Medical University, Moriguchi, Osaka, Japan.
Omori
Kyoko
K
Omori
Koichiro
K
Otani
Hitomi
H
Suzukawa
Junko
J
Inagaki
Chiyoko
C
D000641:Ammonia / Q000633:toxicity*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D002118:Calcium / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism*; D018755:GABA Agonists / Q000494:pharmacology; D006624:Hippocampus / Q000166:cytology; D009474:Neurons / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D010766:Phosphorylation; D051381:Rats; D017208:Rats, Wistar; D018079:Receptors, GABA / Q000187:drug effects*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D051025:bcl-Associated Death Protein
C494821:Bad protein, rat; D002352:Carrier Proteins; D018755:GABA Agonists; C087200:GABA-C receptor; D018079:Receptors, GABA; D051025:bcl-Associated Death Protein; D000641:Ammonia; D002118:Calcium