Ammonia induces rapid clustering and membrane translocation of GLAST in cultured rat astrocytes

Background Glutamate homeostasis is disturbed in hepatic encephalopathy (HE) and is thought to play a role in its pathogenesis (1). In cultured rat astrocytes, glutamate is released as a consequence of exposure to ammonia in a calcium-dependent mechanism (2). We observed that short time incubation of cultured rat astrocytes transfected with eYFP-GLAST with ammonia (5mmol/l) induced the formation of GLAST clusters.

Aim Our study aims to improve the understanding of the alteration of glutamate homeostasis in HE by investigating GLAST clustering, membrane translocation and multimerization.

Methods Astrocytes, isolated from cerebral cortex of Wistar rats, were transfected with an eYFP tagged GLAST or/and a mCherry tagged GLAST. Using living cells, Total Internal Reflection Microscopy (TIRFM) was performed to investigate the alteration of localization of eYFP-GLAST-molecules upon addition of ammonia. Multimerization of GLAST was investigated using Fluorescence Resonance Energy Transfer-Fluorescence Life Time microscopy (FRET-FLIM) by looking at variations of fluorescence lifetime and fluorescence anisotropy of eYFP-GLAST/GLAST-eYFP before and after exposition of cultured rat astrocytes to ammonia.

Results Using TIRF microscopy, a statistically significant increase of eYFP-GLAST/GLAST-eYFP clusters was observed at the cell surface after 30 seconds stimulation of cultured rat astrocytes with ammonia (5mmol/l) (n = 4). Furthermore preliminary data from FRET-FLIM microscopy experiments indicated a decrease in fluorescence lifetime and an increase in anisotropy of eYFP-GLAST/GLAST-eYFP in cells expressing eYFP-GLAST/mCherry-GLAST and GLAST- eYFP/GLAST-mCherry upon exposure to ammonia.

Conclusion TIRF microscopy results suggest a translocation of eYFP-GLAST/GLAST-eYFP from the cytoplasm to the cell membrane during GLAST clustering and first FRET-FLIM data indicate a GLAST multimerization upon exposure of cultured rat astrocytes to ammonia.

• (1) Schmidt W et al. (1990) Hepatic encephalopathy influences high-affinity uptake of transmitter glutamate and aspartate into the hippocampal MetabBrain Dis. 5:19-31

• (2) Görg B et al. (2010) Ammonia triggers exocytotic release of L-glutamate from cultured rat astrocytes. Glia. 58:691-705

Funded by the research commission of the medical faculty of Heinrich-Heine University Düsseldorf (reference 2018-14) and by the DFG through SFB 974: “Communication and System Relevance in Liver Injury and Regeneration”.

Publication History

Article published online:
04 January 2021