eEF-2 Phosphorylation Down-Regulates P-Glycoprotein Over-Expression in Rat Brain Microvessel Endothelial Cells

• Published in: PloS one Vol. 10; no. 5; p. e0125389
• Main Authors: Tang, Xing Hua, Wu, Xun Yi, Xu, Lan, Fang, You Xin, Wang, Ping, Zhu, Guo Xing, Hong, Zhen
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 11.05.2015; Public Library of Science (PLoS)
• Subjects: Animals; ATP-Binding Cassette, Sub-Family B, Member 1 - genetics; ATP-Binding Cassette, Sub-Family B, Member 1 - metabolism; Blood-brain barrier; Brain; Brain - blood supply; Brain cancer; Cell culture; Cells, Cultured; Down-Regulation; Elongation; Elongation Factor 2 Kinase - antagonists & inhibitors; Elongation Factor 2 Kinase - metabolism; Endothelial cells; Endothelial Cells - metabolism; Endothelium, Vascular - cytology; Endothelium, Vascular - metabolism; Gene expression; Glutamic acid; Glutamic acid receptors (ionotropic); Glycoproteins; Immunohistochemistry; Inhibitors; Kinases; Laboratory animals; Meninges; Microvessels - cytology; Multidrug resistance; N-Methyl-D-aspartic acid receptors; Neurology; Overexpression; P-Glycoprotein; Peptides; Phosphorylation; Polymerase chain reaction; Prostate cancer; Protein synthesis; Proteins; Rats; Rats, Wistar; Receptors; Rodents; Studies
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0125389

We investigated whether glutamate, NMDA receptors, and eukaryote elongation factor-2 kinase (eEF-2K)/eEF-2 regulate P-glycoprotein expression, and the effects of the eEF-2K inhibitor NH125 on the expression of P-glycoprotein in rat brain microvessel endothelial cells (RBMECs). Cortex was obtained from newborn Wistar rat brains. After surface vessels and meninges were removed, the pellet containing microvessels was resuspended and incubated at 37°C in culture medium. Cell viability was assessed by the MTT assay. RBMECs were identified by immunohistochemistry with anti-vWF. P-glycoprotein, phospho-eEF-2, and eEF-2 expression were determined by western blot analysis. Mdr1a gene expression was analyzed by RT-PCR. Mdr1a mRNA, P-glycoprotein and phospho-eEF-2 expression increased in L-glutamate stimulated RBMECs. P-glycoprotein and phospho-eEF-2 expression were down-regulated after NH125 treatment in L-glutamate stimulated RBMECs. eEF-2K/eEF-2 should have played an important role in the regulation of P-glycoprotein expression in RBMECs. eEF-2K inhibitor NH125 could serve as an efficacious anti-multidrug resistant agent.