Glutamate up-regulates P-glycoprotein expression in rat brain microvessel endothelial cells by an NMDA receptor-mediated mechanism
Introduction
The blood-brain barrier (BBB) is formed by the tight junction that connects the brain microvessel endothelial cells (BMECs), thus restricting the entry of compounds from the circulating blood to the brain via paracellular route. For hydrophilic compounds, therefore, the entry into the brain is restricted by the BBB. In addition, the brain uptake of some lipophilic compounds, such as cyclosprin A, doxorubicin, vincristine, has been reported to be restricted. Recent studies have shown that P-glycoprotein (P-gp), which confers multi-drug resistance (MDR) to tumor cells, is located on the luminal membrane of BMECs and mediates active P-gp substrates, including cyclosprin A, doxorubicin, vincristine, efflux into systemic circulation Schinkel et al., 1994, Sakata et al., 1994, Tsuji et al., 1992. Therefore, P-gp plays an important role in the integrity of BBB and protects the brain from many exogenous toxins and sudden changes in the levels of cerebral transmitters.
During ischemic and anoxic injury, the alteration in the central nervous system (CNS) microenvironment can result in the changes in BBB function. Some transporters in BMECs, such as P-gp, may be altered under these pathological conditions. Production of intracellular reactive oxygen species (ROS) following hypoxia/reoxygenation or H2O2 treatment is known to lead to the increase of P-gp expression in rat brain microvessel endothelial cells (RBMECs) (Felix and Barrand, 2002).
Glutamate, a principal excitatory neurotransmitter in the mammalian CNS, has been shown to be involved in the pathogenesis of many CNS disorders Choi, 1998, Coyle and Puttfarcken, 1993. ROS generated through activation of NMDA receptor is believed to be an important factor in the toxicity of glutamate. In the recent studies, the presence of NMDA receptor has been confirmed in BMECs by using reverse transcriptase-polymerase chain reaction (RT-PCR) St'astny et al., 2002, Krizbai et al., 1998. It has been demonstrated that activation of the NMDA receptor may cause breakdown of the barrier (Koenig et al., 1992). However, the role of glutamate receptors under physiological and pathological conditions of the BBB is still unclear. Little is known about the possible influence of glutamate on the transporters, including P-gp, in the BBB. The present study was undertaken to investigate the effect of glutamate on the expression and activity of P-gp in RBMECs and its mechanism.
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Materials
P-gp monoclonal antibody (MRK16, C219) was a product of Kamiya Biomedical Co. (Seattle, WA, USA). Fluorescein isothiocyanate (FITC) labeled goat anti-rabbit IgG was purchased from Sino-American Biotechnology Co. (Shanghai, China). Rhodamine 123 (Rh123), L-glutamate, MK801 and N-acetylcysteine were obtained from Sigma Chemical Co. (St. Louis, MO, USA). All other chemicals were of analytical grade and commercially available.
Isolation of rat brain microvessel endothelial cells
Endothelial cells were isolated from rat brain according to the modified
Glutamate induced cytotoxicity of RBMECs
The cytotoxic effect of glutamate is shown in Fig. 1. After RBMECs were exposed to 300, 1000, 3000 μM glutamate for 30min and followed by incubation for 48h, the relative cell viability was 87.8%, 72.5% and 54.5%, respectively. Cells damage induced by 3000 μM glutamate could be prevented by the pre-treatment with 100 μM noncompetitive NMDA antagonist MK801. No cytotoxic effect of glutamate at a concentration of 100 μM was observed. Therefore all further investigations on the effects of
Discussion
This study was undertaken to investigate the possible effects of glutamate on P-gp expression and activity in RBMECs that comprise the BBB. Activation of glutamate receptors has been shown to mediate a large number of neuronal processes such as ischemic and anoxic damage. In addition to neurons and glia, glutamate receptors, including NMDA receptor, have been demonstrated in BMECs St'astny et al., 2002, Krizbai et al., 1998. It has been shown that activation of glutamate receptors may cause
Acknowledgements
This work was supported by the National Nature Science Foundation of China, No 39870885.
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