Therefore, during liver disease, the loss of hepatic function leads to hyperammonemia and consequently increased brain ammonia and hepatic encephalopathy (HE). Reducing ammonia neurotoxicity through systemic ammonia-lowering strategies remains the mainstay therapeutic strategy for HE. Ammonia, both as an ion (NH4+) and gas (NH3), easily crosses all plasma membranes, including the blood brain barrier (BBB); the interface between the blood and the brain. Glutamine synthetase (GS), an enzyme which during the process of amidating glutamate to glutamine removes ammonia, plays an important compensatory role during liver disease. GS is expressed in muscle and brain (astrocytes) but has never been thoroughly explored in the endothelial cells of the BBB.
Methods: Using primary rat brain microvascular endothelial cells (ECs), the presence of GS was assessed using rtPCR, western blot, immunohistochemistry and activity assay. Furthermore, we isolated cerebral microvessels (CMV) from the frontal cortex of naïve rats and measured GS activity and protein expression (brain lysate was used as positive control). GS was also evaluated by immunohistochemistry (co-localized with caveolin-1 (marker for ECs). In addition, GS activity was assessed in ECs exposed to 1mM of ammonium chloride for 48h. Finally, GS activity and protein expression were evaluated in ECs submitted to plasma from 6-week bile-duct ligated (BDL) rats or sham-operated controls for 72h.
Results: ECs expressed mRNA, protein and activity of GS. However, EC`s expression (normalized to g/protein) of GS was lower compared to brain lysate control samples (p<0.05). GS protein expression in CMV showed similar results to that of brain but GS activity was significantly less in CMV (p<0.05). Using immunohistochemistry, GS was detected in ECs and in CMV from naïve rats. When ECs were exposed to ammonia (1mM), an increase in GS activity was demonstrated (p<0.05). However, when exposed to conditioned medium from BDL rats, GS activity and protein expression were decreased when compared to sham-operated controls (p<0.05).
Discussion: These results demonstrate for the first time that GS is present in ECs in both in vivo and in vitro. The lower expression of the enzyme in CMVs compared to that found in the brain, could reason why GS has never been reported in these cells. Interestingly, ammonia stimulates GS activity in ECs, but GS activity is decreased following treatment with plasma from BDL rats. This suggests other factors such as oxidative stress and inflammation, could inhibit GS activity. We speculate that a downregulation of GS in the BBB allows for a faster entry of ammonia into the brain and therefore may play a significant role in the onset of HE. We anticipate upregulating GS in ECs of the BBB could become a new therapeutic target for HE.
