Effect of ammonia on ciliary neurotrophic factor mRNA and protein expression and its upstream signalling pathway in cultured rat astroglial cells: possible implication of c-fos, Sp1 and p38MAPK.
Ciliary neurotrophic factor (CNTF) may be implicated in the pathogenetic mechanisms of hepatic encephalopathy. We tested this hypothesis by treating confluent primary cultures of rat astroglial cells with ammonium chloride for various periods and analysing the effect of ammonia on the signalling pathway that regulates CNTF mRNA and protein expression. Ammonia treatment induced a dose- and time-dependent reduction in CNTF mRNA and protein expression. Surface-enhanced laser desorption/ionization-time-of-flight mass spectrometry analysis of CNTF in the culture medium demonstrated that ammonia also induced a significant decrease in CNTF release. In addition, ammonia affected Sp1 and c-fos, transcription factors that regulate CNTF mRNA and protein expression, which showed partial dephosphorylation and significantly lower mRNA and protein levels. Total content of p38MAPK (for which Sp1 and c-fos are substrates) was unaffected by ammonia, although the diphosphorylated (active) form was significantly reduced after ammonia exposure.
Bodega
G
G
Departamento de Biología Celular y Genética, Facultad de Biología, Universidad de Alcalá, E-28871 Alcalá de Henares, Madrid, Spain. guillermo.bodega@uah.es
Suárez
I
I
Almonacid
L
L
Ciordia
S
S
Beloso
A
A
López-Fernández
L A
LA
Zaballos
A
A
Fernández
B
B
D000641:Ammonia / Q000494:pharmacology*; D000818:Animals; D001253:Astrocytes / Q000187:drug effects / Q000378:metabolism*; D015153:Blotting, Western; D002478:Cells, Cultured; D020934:Ciliary Neurotrophic Factor / Q000096:biosynthesis*; D015180:Electrophoresis, Gel, Two-Dimensional; D016762:Genes, fos / Q000187:drug effects*; D010766:Phosphorylation; D012333:RNA, Messenger / Q000096:biosynthesis*; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects*; D016329:Sp1 Transcription Factor / Q000096:biosynthesis* / Q000187:drug effects; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D048051:p38 Mitogen-Activated Protein Kinases / Q000096:biosynthesis* / Q000187:drug effects
D020934:Ciliary Neurotrophic Factor; D012333:RNA, Messenger; D016329:Sp1 Transcription Factor; D000641:Ammonia; D048051:p38 Mitogen-Activated Protein Kinases