MODULATION OF TIGHT JUNCTION STRUCTURE IN BLOOD-BRAIN-BARRIER ENDOTHELIAL-CELLS - EFFECTS OF TISSUE-CULTURE, 2ND MESSENGERS AND COCULTURED ASTROCYTES

Tight junctions between endothelial cells of brain capillaries are the most important structural elements of the blood-brain barrier. Cultur ed brain endothelial cells are known to loose tight junction-dependent blood-brain barrier characteristics such as macromolecular impermeabi lity and high electrical resistance. We have directly analyzed the str ucture and function of tight junctions in primary cultures of bovine b rain endothelial cells using quantitative freeze-fracture electron mic roscopy, and ion and inulin permeability. The complexity of tight junc tions, defined as the number of branch points per unit length of tight junctional strands, decreased 5 hours after culture but thereafter re mained almost constant. In contrast, the association of tight junction particles with the cytoplasmic leaflet of the endothelial membrane bi layer (P-face) decreased continuously with a major drop between 16 hou rs and 24 hours. The complexity of tight junctions could be increased by elevation of intracellular cAMP levels while phorbol esters had the opposite effect. On the other hand, the P-face association of tight j unction particles was enhanced by elevation of cAMP levels and by cocu lture of endothelial cells with astrocytes or exposure to astrocyte-co nditioned medium. The latter effect on P-face association was induced by astrocytes but not fibroblasts. Elevation of cAMP levels together w ith astrocyte-conditioned medium synergistically increased transendoth elial electrical resistance and decreased inulin permeability of prima ry cultures, thus confirming the effects on tight junction structure a nd barrier function. P-face association of tight junction particles in brain endothelial cells may therefore be a critical feature of blood- brain barrier function that can be specifically modulated by astrocyte s and cAMP levels. Our results suggest an important functional role fo r the cytoplasmic anchorage of tight junction particles for brain endo thelial barrier function in particular and probably paracellular perme ability in general.