New insights on ammonia metabolism in endothelial cells of the blood brain barrier
Hepatic encephalopathy (HE) is a complex neuropsychiatric syndrome, which is a major complication of liver disease. Cytotoxic edema is a serious complication of HE, known to be the result of astrocyte swelling. Blood derived pathogenic factors such as ammonia (NH4+) and oxidative stress’ (OS) are known to be synergistically implicated. Endothelial cells (EC) of the blood brain barrier (BBB) are the first cells regulating passage into the brain and to contact blood-derived molecules. The effect of ammonia and oxidative stress on EC transport and metabolism has never been thoroughly explored. Therefore, our aim was to evaluate the effects of NH4+ and reactive oxygen species (ROS) on EC of the BBB using in vivo and in vitro models systems. The Na–K–2Cl cotransporter (NKCC1) has been demonstrated to be involved in the pathogenesis of brain edema in numerous neurological conditions. NKCC1 can transport NH4+ into the brain and is regulated by ROS. Therefore, the expression of NKCC1 was evaluated in primary EC submitted to different concentrations of ROS and NH4+ as well as in cerebral microvessels (CMV) isolated from the bile-duct ligated (BDL) rat, a model HE induced by chronic liver disease. No upregulation of NKCC1 was present in either the treated EC or CMV. Glutamine synthetase (GS) is an enzyme with an important compensatory role in NH4+ detoxification during liver disease. GS is expressed in muscle and brain (astrocytes) but has never been thoroughly explored in ECs of the BBB. GS protein expression and activity was found in EC of the BBB in vitro (primary EC) and in vivo (CMV isolated from naive rats). In the BDL model, GS expression in CMVs was not significantly different from SHAM-operated controls. In addition, we treated ECs with conditioned medium from plasma of BDL rats and found a decrease in GS protein and activity when compared to SHAM. Furthermore, EC treated with NH4+ increased GS activity while treatments with ROS with and without NH4+ decreased GS activity. Overall these results demonstrate for the first time that GS is present in EC both in vivo and in vitro. GS is downregulated in EC treated with BDL plasma (but not in BDL CMV). Interestingly, NH4+ stimulates GS activity in ECs, while ROS inhibits GS activity, possibly justifying the results found from the conditioned medium studies. We speculate that ROS prevents the upregulation of GS in the BBB, decreasing the capacity of the EC to detoxify ammonia and to limit ammonia entry into the brain. We foresee that upregulating GS in ECs of the BBB could become a new therapeutic target for HE.
Hepatic encephalopathy Hyperammonemia Endothelial cells Brain edema Glutamine synthetase NKCC1