(NO)-N-center dot and oxyradical metabolism in new cell lines of rat braincapillary endothelial cells forming the blood-brain barrier

To investigate the relevance of (NO)-N-circle and oxyradicals in the blood- brain barrier (BBB), differentiated and well-proliferating brain capillary endothelial cells (BCEC) are required. Therefore, rat BCEC (rBCEC) were tra nsfected with immortalizing genes. The resulting lines exhibited endothelia l characteristics (factor VIII, angiotensin-converting enzyme, high prostac yclin/thromboxane release rates) and BBB markers (,gamma -glutamyl transpep tidase, alkaline phosphatase). The control line rBCEC2 (mock transfected) r evealed fibroblastoid morphology, less factor VIII, reduced gamma -glutamyl transpeptidase, weak radical defence, low prostanoid metabolism, and limit ed proliferation. Lines transfected with immortalizing genes (especially rB CEC4, polyoma virus large T antigen) conserved primary properties: epithelo id morphology, subcultivation with high proliferation rate under pure cultu re conditions, and powerful defence against reactive oxygen species (Mn-, C u/Zn-superoxide dismutase, catalase, glutathione peroxidase, glutathione) e ffectively controlling radical metabolism. Only 100 muM H2O2 overcame this defence and stimulated the formation of eicosanoids similarly as in primary cells. Some BBB markers were expressed to a lower degree; however, coculti vation with astrocytes intensified these markers (e.g., alkaline phosphatas e) and paraendothelial tightness, indicating induction of BBB properties. I nducible NO synthase was induced by a cytokine plus lipopolysaccharide mixt ure in all lines and primary cells, resulting in (NO)-N-circle release. Com paring the cell lines obtained, rBCEC4 are stable immortalized and reveal t he best conservation of properties from primary cells, including enzymes pr oducing or decomposing reactive species. These cells can be subcultivated i n large amounts and, hence, they are suitable to study the role of radical metabolism in the BBB and in the cerebral microvasculature. (C) 2001 Academ ic Press.