Effect of HE-relevant factors on GLAST clustering in cultured rat astrocytes

Background:

Hepatic encephalopathy (HE) is a neuropsychiatric syndrome frequently accompanying liver failure. Currently, HE is seen as a clinical manifestation of a low grade cerebral edema and cerebral oxidative stress. HE is triggered by a heterogeneous set of factors which action integrates at the level of inducing osmotic and oxidative stress in astrocytes. Several studies have shown that glutamatergic neurotransmission in the brain is disturbed in HE. Glutamate is the main excitatory neurotransmitter in the brain and thus the extracellular glutamate concentrations are tightly controlled by two sodium dependent glutamate transporters: Glutamate transporter 1 (GLT-1) and glutamate aspartate transporter (GLAST). Recent studies showed that ammonia as well as astrocyte swelling per se induce the rapid release of glutamate by a calcium-dependent vesicular release mechanism in astrocytes. Long term treatment of cultured astrocytes with ammonia (>= 48h) impairs glutamate uptake through downregulation of GLAST which may be a consequence of GLAST mRNA oxidation. Furthermore short term incubation with ammonia also impairs glutamate uptake in astrocytes in a sodium and ph-dependent way.

Aims and Methods:

Our study focuses on the rapid effects of ammonia on the intracellular GLAST localisation. Therefore we transfected cultured rat astrocytes with a vector coding for a YFP-tagged GLAST.

Results:

By using conventional epifluorescence microscopy an almost instantaneous clustering of GLAST-YFP molecules after exposure to NH4Cl (5 mmol/l) was detected. Also lower concentrations of NH4Cl down to 0,5 mmol/l were able to induce the clustering. A similar effect was seen after exposure to glutamate (100 µmol/l). CH3NH3Cl which causes a similar pH-change as NH4Cl did not lead to a clustering of the YFP-tagged GLAST molecules in cultured rat astrocytes. Further examinations showed that the clustering of GLAST is Ca2+ and NO dependant as it can be prevented by using the Ca-chelator BAPTA (10 µmol/l) or the inhibitor of NO-Synthase L-NAME (1 mmol/l) or induced by the NO-donor DEANONOate (50 µmol/l).

Conclusions and Outlook:

Our data suggest that in cultured rat astrocytes GLAST clusters rapidly after exposure to NH4Cl in an Ca2+ and NO dependent manner. In further studies we will closer analyse the lokalisation of the GLAST clustering using TIRF-microscopy and the possible multimerisation of GLAST using FRET-microscopy.