title: Ammonium Increases TRPC1 Expression Via Cav-1/PTEN/AKT/GSK3β Pathway.
abstract: Hyperammonemia occurring following acute liver failure is the primary cause of hepatic encephalopathy. In the brain, ammonium is catabolised by glutamine synthetase expressed exclusively in astroglia; ammonium overload impairs astroglial homeostatic systems. Previously, we had reported that chronic treatment with 3 mM ammonia increased expression of transient receptor potential canonic 1 (TRPC1) channels and Ca2+release from intracellular Ca2+stores (Liang et al. in Neurochem Res 39:2127-2135, 2014). Glycogen synthase kinase 3β (GSK-3β) has a key role in several astroglial signalling pathways and is known to be affected in various CNS diseases. We have studied the involvement of Cav-1/PTEN/AKT/GSK-3β signalling system in regulation of TRPC1 gene expression by ammonium. Effects of chronic (1-5 days) treatment with ammonium chloride (ammonium), at pathologically relevant concentrations of 1-5 mM were investigated on primary cultures of mouse cerebral astrocytes. We quantified expression of caveolin-1 (Cav-1), membrane content of phosphatase and tensin homologue (PTEN), phosphorylation of AKT and GSK-3β, and expression of TRPC1 channels. Ammonium significantly increased expression of Cav-1 mRNA and protein, mRNA of TRPC1 as well as membrane content of PTEN; conversely phosphorylation of AKT and GSK-3β were significantly decreased. These changes were abolished following astrocytes treatment with siRNA specific to Cav-1, indicating the involvement of Cav-1/PTEN/PI3K/AKT pathway. Similar results were found in the brains of adult mice subjected to intraperitoneal injection of urease (a model for hyperammoniemia) for 1-5 days. In transgenic mice tagged with an astrocyte-specific or neurone-specific markers (used for fluorescence-activated cell sorting of astrocytes vs. neurones) and treated with intraperitoneal injections of urease for 3 days, the Cav-1 gene mRNA expression was up-regulated in astrocytes, but not in neurones. The up-regulation of TRPC1 gene expression by ammonium was suppressed by GSK-3β inhibitors, lithium salt and AR-A014418, suggesting that increase of GSK-3β activity may play a role in ammonium-related pathologies.
authors.affiliation: Laboratory of Metabolic Brain Diseases, Institute of Metabolic Disease Research and Drug Development, China Medical University, No. 77, Puhe Road, Shenbei District, Shenyang, People's Republic of China.
authors.forename: Wei
authors.initials: W
authors.lastname: Wang
authors.affiliation: Laboratory of Metabolic Brain Diseases, Institute of Metabolic Disease Research and Drug Development, China Medical University, No. 77, Puhe Road, Shenbei District, Shenyang, People's Republic of China.
authors.forename: Li
authors.initials: L
authors.lastname: Gu
authors.affiliation: Faculty of Life Science, The University of Manchester, Manchester, UK.|Achucarro Center for Neuroscience, IKERBASQUE, Basque Foundation for Science, 48011, Bilbao, Spain.|University of Nizhny Novgorod, Nizhny Novgorod, 603022, Russia.
authors.forename: Alexei
authors.initials: A
authors.lastname: Verkhratsky
authors.affiliation: Laboratory of Metabolic Brain Diseases, Institute of Metabolic Disease Research and Drug Development, China Medical University, No. 77, Puhe Road, Shenbei District, Shenyang, People's Republic of China. hkkid08@yahoo.com.
authors.forename: Liang
authors.initials: L
authors.lastname: Peng
mesh_terms: D000641:Ammonia / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D001253:Astrocytes / Q000378:metabolism; D051242:Caveolin 1 / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D005260:Female; D000071679:Glycogen Synthase Kinase 3 beta / Q000037:antagonists & inhibitors / Q000378:metabolism*; D022124:Hyperammonemia / Q000378:metabolism; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D051059:PTEN Phosphohydrolase / Q000378:metabolism*; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics; D050052:TRPC Cation Channels / Q000378:metabolism*
chemical_list: C495541:Cav1 protein, mouse; D051242:Caveolin 1; D034741:RNA, Small Interfering; D050052:TRPC Cation Channels; C096351:transient receptor potential cation channel, subfamily C, member 1; D000641:Ammonia; D000071679:Glycogen Synthase Kinase 3 beta; D051057:Proto-Oncogene Proteins c-akt; D051059:PTEN Phosphohydrolase; C487757:Pten protein, mouse
keywords: Ammonium; Astrocyte; Cav-1, PTEN, PI3K/AKT; GSK-3β; Hepatic encephalopathy