Effects of serine protease inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF) on the blood-brain barrier (BBB) functions of cultured cerebral microvascular endothelial cells (CMEC)

P Megyeri MD, PhDa, MA Deli MD, PhDb,c, H Takahata MDc, M Niwa MD, PhDc, KM Pabst BScd, MJ Pabst PhDd, CS Ábrahám MDc

aDepartment of Pediatrics and Infectious Diseases, University of Szeged, Albert Szent-Györgyi School of Medicine, Szeged, Hungary.bLaboratory of Molecular Neurobiology, Institute of Biophysics, Biological Research Center, Hungarian Academy of Sciences, Szeged, Hungary.cDepartment of Pharmacology, Nagasaki University School of Medicine, Nagasaki, Japan. dDental Research Center and Department of Biochemistry, University of Tennessee, Memphis, TN USA.

BACKGROUND: The BBB restricts free passage of solutes and cells, provides a special compartment for brain by transport of nutrients, and protects brain from drugs by efflux systems. AIM: Reveal the effects of AEBSF, a water-soluble, irreversible inhibitor of serine proteases, on BBB functions in vitro. METHODS: Cultures of primary CMEC derived from newborn pigs and rats, and GP8 rat brain endothelial cell line were used. Viability study was performed using MTT reduction and LDH release assays. Non-specific transport was determined by Lucifer yellow uptake, scavenger receptor-mediated binding and uptake by DiI-labeled acetylated low density lipoprotein (DiI-AcLDL). Superoxide release was measured by cytochrome c reduction, while P-glycoprotein activity by rhodamine 123 assay. RESULTS: AEBSF, in doses of 50-100-200 µM, did not change the viability of CMEC up to 24 h, while concentrations of 1 and 2 mM were already toxic after 3 h. Rate of fluid-phase endocytosis was unchanged after 3 h pre-treatment with non-toxic, lower doses of AEBSF, although 2 mM AEBSF for 3 h caused a huge increase in Lucifer yellow uptake. AEBSF did not change DiI-AcLDL binding, but decreased the uptake in doses higher than 50 µM. A dose-dependent decrease in superoxide release induced by hypoxia-reoxygenation was also seen after AEBSF treatment. Co-incubation with 1 and 2 mM AEBSF for 30 min significantly inhibited the activity of P-glycoprotein, while lower doses were ineffective. CONCLUSION: Serine protease inhibitor AEBSF regulates BBB functions and may contribute to the development of effective therapeutical approaches aiming intracerebral malignancies.

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Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Paris, France; February 9 - 12, 2002; in the section on Novel Therapies, Part 1.