γ-aminbuturic acid A receptor mitigates homocysteine-induced endothelial cell permeability

Many cerebrovascular disorders are accompanied by an increased homocysteine (Hey) levels. We have previously shown that acute hyperhomocysteinemia (HHcy) leads to an increased microvascular permeability in the mouse brain. Hey competitively binds to gamma-aminbuturic acid (GABA) receptors and may increase vascular permeability by acting as an excitatory neurotransmitter. However, the role of GABA-A (GABA(A)) receptor in Hey-induced endothelial cell (EC) permeability remains unclear. In the present study we attempted to determine the role of GABAA receptor and the possible mechanisms involved Hcy-induced EC layer permeability. Mouse aortic and brain ECs were grown in Transwells and treated with 50 mu M Hey in the presence or absence of GABA(A)-specific agonist muscimol. Role of matrix metalloproteinase-9 (MMP-9) was determined using its activity inhibitor GM-6001. Involvement of extracellular signal-regulated kinase (ERK) signaling was assessed using its kinase activity inhibitors PD98059 or U0126. EC permeability to the known content of bovine serum albumin (BSA)-conjugated with Alexa Flour-488 was assessed by measuring fluorescence intensity of the solutes in the Transwell's lower chambers. It was found that Hey induced the formation of filamentous actin (F-actin). Hey-induced EC permeability to BSA was significantly decreased by GABA and muscimol treatments. Presence of MMP-9 or ERK kinase activity inhibitors restored the Hcy-induced EC permeability to its baseline level. The mediation BSA leakage through the ECs was further confirmed in the experiments where Hcy-induced alterations in transendothelial electrical resistance of confluent ECs were assessed. The data suggest that Hey increases EC layer permeability through inhibition of GABAA receptor and F-actin formation, in part, by transducing ERK and MMP-9 activation.