1. ¹Molecular Neuroscience and Vascular Biology Laboratory, Department of Surgery, University of Kentucky Medical Center, Lexington, Kentucky, USA
2. ²Biological Research Center, Szeged, Hungary
3. ³College of Agriculture, University of Kentucky, Lexington, Kentucky, USA

Correspondence: Dr M Toborek, Molecular Neuroscience and Vascular Biology Laboratory, Department of Surgery/Neurosurgery, University of Kentucky Medical Center, 800 Rose Street, Lexington, Kentucky 40536, USA. E-mail: michal.toborek@uky.edu

Received 8 March 2006; Revised 11 September 2006; Accepted 14 November 2006; Published online 24 January 2007.

Abstract

Glutamate levels increase dramatically in cerebral ischemia and stroke. This may lead to opening of the blood–brain barrier (BBB) and induce further brain damage. Because endothelial tight junctions are critical elements of the BBB integrity, the aim of this study was to investigate the mechanisms of glutamate-induced alterations of the tight-junction protein occludin in cultured brain microvascular endothelial cells (BMECs). Transient exposure to glutamate resulted in cellular redistribution of occludin, followed by a decrease in the total level of this protein and diminished barrier function of BMECs. Inhibition of the N-methyl-D-aspartate (NMDA) or alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate (AMPA/KA) receptors attenuated glutamate-induced changes in occludin redistribution but not in the total protein levels. Treatment with glutamate also increased tyrosine phosphorylation and decreased threonine phosphorylation of occludin. Inhibition of the NMDA receptors by MK-801 partially protected against glutamate-induced elevation of occludin tyrosine phosphorylation. In addition, pretreatment with MK-801-attenuated glutamate-mediated disruption of endothelial barrier function. Blocking of the AMPA/KA receptors by 6,7-dinitroquinoxaline-2.3-dione (DNQX) protected against hypophosphorylation of threonine residues of occludin; however, it did not affect disruption of endothelial integrity. These findings indicate the opposite effects of the NMDA and AMPA/KA receptors on occludin phosphorylation and disruption of the BBB functions.