Elsevier

Neuropharmacology

Volume 43, Issue 6, November 2002, Pages 1006-1014
Neuropharmacology

Nitronyl nitroxides, a novel group of protective agents against oxidative stress in endothelial cells forming the blood−brain barrier

https://doi.org/10.1016/S0028-3908(02)00180-6Get rights and content

Abstract

Nitronyl nitroxides (NN) effectively decompose free radicals (Haseloff, Zoellner, Kirilijuk, Grigoriev, Reszka, Bernhardt, Mertsch, Roloff and Blasig, 1997a Free Radical Research 26, 7–17). As brain endothelium, forming the blood-brain barrier (BBB), is both the main source and the target of reactive species during cerebral oxidative stress, we studied the effect of NN on brain endothelial cells injured by the mediator of oxidative stress H2O2 (Kondo, Kinouchi, Kawase and Yoshimoto, 1996. Neuroscience Letters 215, 103–106). H2O2 caused hydroxyl radical generation, lipid peroxidation, membrane dysfunction, membrane leak and cell death, concentration dependently. Due to 0.5 mM H2O2, oxy-radical-induced membrane phospholipid peroxidation (malondialdehyde) increased to 0.61±0.04 nmol/mg protein vs control (0.32±0.03, p<0.05), cells lost cytosolic proteins into the medium and viability decreased to 28±2% of control (p<0.05). Permeability through the endothelial monolayer (measure for the tightness of the BBB) rose to 250±40% after 0.15 mM H2O2 (p<0.001). Addition of 10 μM of the NN 5,5-dimethyl-2,4-diphenyl-4-methoxy-2-imidazoline-3-oxide-1-oxyl (NN-2), 1 mM phenylbutyl nitrone (PBN), or 10 μM of the lazaroid U83836E improved cell viability during incubation with 0.5 mM H2O2 to 57±1%, 49±2%, and 42±3% (p<0.05, vs drug-free H2O2 group). The permeability enhancement by 0.15 mM H2O2 was reduced to 171±21%, 170±25%, and 118±32% (p<0.05 vs drug-free H2O2 group). Generally, the assumption is supported that during cerebral oxidative stress the protection should also be directed to the cells of the BBB, which can be provided by antioxidative approaches. NN represent a new group of antioxdatively acting cytoprotectiva improving the survival and function of the endothelium against oxidative stress.

Introduction

Recently it has been shown that antioxidants and radical scavengers, such as phenylbutyl nitrone (PBN) and lazaroids may protect the brain against oxidative stress induced by ischemia (Hall et al., 1991, Langley et al., 2000), hypoxia (Rauca et al., 2000) or inflammation (Haddad et al., 1998, Park et al., 2000). These studies focus on neuroprotection. In contrast, the blood–brain barrier (BBB) formed by brain endothelial cells (BEC) is neglected with respect to pharmacological and antioxidative approaches in brain injury. This is surprising as the endothelium is considered a main source of reactive species (Kondo et al., 1996, Terada, 1996) and as a primary target for the damaging action of reactive species (Schulz et al., 1997).

BEC are highly susceptible to oxidative stress (Mertsch et al., 1995), and hydrogen peroxide (H2O2) is an important mediator of oxidative cell injury (Horwitz et al., 1996). Hypoxia/reoxygenation cause opening of the BBB (Giese et al., 1995) and endothelial release of H2O2 (Kondo et al., 1996), as well as of superoxide, (O2; Terada, 1996) decomposed to H2O2 by superoxide dismutase. Adhesion of neutrophils to arterial endothelial cells following ischemia amplifies oxidative stress via formation of the reactive oxygen species (ROS) H2O2, .OH and O2 (Siflinger-Birnboim and Malik, 1993, von Asmuth and Buurman, 1995. BEC possess powerful activity of catalase and glutathione peroxidase detoxifying H2O2 (Schroeter et al., 1999). Upon hypoxia, activity of defense enzymes is reduced (Plateel et al., 1995) and can be overwhelmed by high amounts of ROS (Utepbergenov et al., 1998, Schroeter et al., 1999).

Nitronyl nitroxides (NN) have been applied to detect nitric oxide (NO) (Akaike et al., 1993, Haseloff et al., 1997a) and to prevent inflammatory diseases accompanied by excessive production of NO (Maeda et al., 1994). Compared to the reaction of NN with NO, NN react with higher rate constants with oxygen free radicals (Haseloff et al., 1997a). Therefore, we hypothesize that NN may reveal cytoprotective effects under conditions with an excess of not only NO but also of ROS and secondary, ROS-derived radicals. Excessive formation of reactive oxygen or nitrogen species can occur during cerebral hypoxia (Rauca et al., 2000), ischemia (Langley et al., 2000) and inflammation (Fujii and Berliner, 1999). However, it is not known whether NN exert protection against ROS in oxidative stress.

The aim of the study was to demonstrate, for the first time, the protective effect of NN compared to cerebroprotective antioxidants (PBN and a lazaroid), using a cell culture model of the BBB injured by H2O2. Our results show that NN represent a new group of cytoprotective agents improving the survival and function of the endothelium under oxidative stress.

Section snippets

Cell cultures

Cloned immortalized rat brain endothelial cells (RBE4, Roux et al., 1994; kindly supplied by Prof. P.-O. Couraud, Paris, France), were cultivated in Ham’s F10/α-medium 1:1 (Biochrom), 10% fetal bovine serum (FBS), 2 mM glutamine, 1 ng basic fibroblast growth factor and 300 μg/ml geneticin (Boehringer Mannheim). Endothelial origin of cells was characterised by phase contrast microscopy (IMT 2, Olympus), by immunostaining for Factor VIII (Dako), alkaline phosphatase (Sigma) and

Radical formation after incubation of endothelial cells with H2O2

After the addition of H2O2 to RBE4 a typical four line signal of the OH adduct of DMPO was registered (line intensity 1:2:2:1, splitting constants aN=aHβ=14.9 mT) (Fig. 1B). The intensity of the OH formation depended on the H2O2 concentration added (Fig. 1A). In the absence of the cells higher signal intensity was registered indicating that a significant amount of the oxy-radicals generated was taken up by the cells (Fig. 1A,B). In the absence of H2O2 no ESR signal was recorded (not shown).

Endothelial cell injury after exposure to H2O2

Fig.

Discussion

H2O2 is noxious to cells causing membrane dysfunction and protein oxidation. After addition of H2O2, LPO may result from the reaction of H2O2 with reduced transition metal ions (or complexes) leading to the formation of OH (Singh et al., 1999). In BEC, we have measured OH formation, membrane LPO and membrane and cell injury, after H2O2 incubation. This is demonstrated by ESR spin trapping, accumulation of MDA (product of oxy-radical induced membrane phospholipid peroxidation), release of

Acknowledgements

This work was supported by BMBF BEO 0311466C, DFG Bl 308/6-1, DFG GK238, DFG SFB 507A2. The authors thank Mrs. Hartmann for support in biochemical analyses and Mrs. Eilemann for technical support in the cell culture lab.

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