Co-culture of the human endothelial-like cell line ECV304 and the rat C6 glioma cell line is a well established model system for the blood-brain barrier. We studied in the tight junction (TJ) composition and changes induced under co-culture conditions. To this end, effects of co-culture on the expression of various TJ proteins was investigated on mRNA (real-time PCR) and protein (Western blot) level. Subcellular distribution of TJ proteins was studied by confocal laser scanning microscopy.

Results: In the absence of C6 glioma cells, ECV304 cells presented a disrupted localization of ZO-1, occludin, and claudin-1 within the TJ area while claudin-3 and -5 appeared to be exclusively localized in the ER. After 6 days of co-culture ZO-1, occludin, claudin-1, -3, and -5 showed a complete TJ pattern. These changes were not mirrored by any changes in the amount of mRNA or protein, indicating that they were due to a redistribution of the proteins within the cells. Redistribution of TJ proteins was accompanied by an increase in transepithelial resistance, Rt, from 32±1 in ECV304 cell layers without co- culture to 56±2 Wcm 2 in the presence of C6 glioma cells. Part of these effects were under PKA control, as dibutyryl-cAMP caused a further increase in Rt, while the PKA inhibitor H8 prevented the Rt increase during co-culture.

Conclusion: Under the influence of C6 glioma cells, BBB-typical claudins 1, 3, and 5 are integrated into TJs of ECV304 cells. Part of this remodelling is controlled by PKA-dependent pathways.