Elsevier

Life Sciences

Volume 75, Issue 11, 30 July 2004, Pages 1313-1322
Life Sciences

Glutamate up-regulates P-glycoprotein expression in rat brain microvessel endothelial cells by an NMDA receptor-mediated mechanism

https://doi.org/10.1016/j.lfs.2004.02.027Get rights and content

Abstract

The accumulation of glutamate in the extracellular space in the central nervous system (CNS) plays a major part in ischemic and anoxic damage. In this study, we examined the effect of glutamate on the expression and activity of P-glycoprotein (P-gp) in rat brain microvessel endothelial cells (RBMECs) making up the blood-brain barrier (BBB). The level of P-gp expression significantly increased in RBMECs after the treatment of 100 μM glutamate. At this concentration, glutamate also enhanced rat mdr1a and mdr1b mRNA levels determined by RT-PCR analysis. Flow cytometry was used to study P-gp activity by analysis of intracellular rhodamine123 (Rh123) accumulation. Overexpression of P-gp resulted in a decreased intracellular accumulation of Rh123 in RBMECs. Glutamate-induced increase of intracellular reactive oxygen species (ROS) was observed by using the 2′,7′-dichlorofluorescein (2′,7′-DCF) assay. MK-801, a non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist, and ROS scavenger N-acetylcysteine obviously blocked ROS generation and attenuated the changes of both expression and activity of P-gp induced by glutamate in RBMECs. These data suggested that glutamate up-regulated P-gp expression in RBMECs by an NMDA receptor-mediated mechanism and that glutamate-induced generation of ROS was linked to the regulation of P-gp expression. Therefore, transport of P-gp substrates in BBB appears to be affected during ischemic and anoxic injury.

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.

Section snippets

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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