Einfluss pathophysiologischer Konzentrationen von Ammonium auf die Transkription und Funktion von Acid-Sensing Ion Channels (ASICs) in murinen Kortexneuronen

Hepatic Encephalopathy is a central neuropsychologic disorder caused by liver failure. An increased ammonium concentration is believed to be one of the main players, resulting in changes in several neurotransmitter systems as well as in energy metabolism, both in neuronal and non-neuronal cells in the brain. This is accompanied by neuroinflammatory processes. These factors act synergistically and lead to worsening of the symptoms. AcidSensing Ion Channels (ASICs) are proton-gated cation channels that belong to the DEG/ENaC gene-family. ASIC1a, ASIC2a and ASIC2b are expressed in the mammalian brain as homo- and heteromeric channels. However, their function has not been conclusively elucidated; they seem to play a role in synaptic transmission as well as in several neuroinflammatory processes (e.g. multiple sclerosis). Besides their activation by acidification they can be modulated by several proinflammatory mediators. It has been shown that high concentration of ammonium (> 20 mM) can directly activate ASICs. This work characterises the effects of pathophysiological ammonium concentrations on ASICs in cortical neurons of mice. Direct effects on channel function, as well as functional aspects caused by cultivating the cells in 5 mM ammonium for several days were analysed using the path-clamp method. Furthermore, changes in the transcription level were investigated by quantitative RT-PCR. As the results show, pathophysiological ammonium concentrations (2 – 4 mM) specifically increase proton sensitivity of homomeric ASIC1a. In addition, cultivation of cortical neurons with ammonium in vitro induces changes on a transcriptional and functional level. The RNA level of cortical ASICs decreases with prolonged ammonium cultivation and thereby seems to change subunit composition towards more ASIC1a on the cell surface. The transcriptional effects are slow compared to other ion channels (e.g. AMPA receptors). Analyzing the RNA level in a mouse model of acute hyperammonemia supports this hypothesis. 24h after NH4-acetate intoxication no change on the transcriptional level of ASICs could be observed. A further aspect is the subunit composition of functional cortical ASICs. Even though data are still ambiguous, ASIC1a/2b could be more abundant as has been assumed so far.- vii Because ASIC1a/2b, like ASIC1a, is capable of inducing acid-induced neuronal cell death, this could make ASIC1a/2b an interesting pharmacological target.